Fluidics devices

ABSTRACT

The invention relates to fluidics as used in medical and diagnostic equipment and relates further to means for purifying, abstracting, filtering, detecting and/or measuring analytes in liquid samples.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/032,356, filed Feb. 15, 2008, entitled the same, and claims thebenefit of U.S. Provisional Patent Applications Nos. 60/890,180, filedFeb. 15, 2007, and 60/953,197, filed Jul. 31, 2007, entitled the same,all of which are incorporated by reference.

FIELD OF THE INVENTION

The invention relates to fluidics as used in medical and diagnosticequipment and relates further to means for purifying, abstracting,filtering, detecting and/or measuring analytes in liquid samples.

BACKGROUND OF THE INVENTION

The following documents are all incorporated herein by reference intheir entireties, although none is admitted to be prior or relevant art.Collectively they reflect that there is a current and long-felt needfor, and past failure of success as relates to, adequate microcassettefabrication and microfluidics, not only from a cost and ease offabrication and reproducibility standpoint, but also from the standpointthat such systems are typically restricted to laminar flow and featureinefficient mixing compounded by gaseous bubble formation that obstructsor restricts flow and diffusion. The present invention, depending onaspect and embodiment, provides useful solutions to one or more of thesehistoric deficiencies.

Woolley et al. (1996) report functional integration of PCR amplificationand capillary electrophoresis in a microfabricated DNA analysis device.Anal. Chem., 68:40814086. The chips are generated by photolithographyand etching of silicone wafers. The prospects for electrophoreticvalving and active microfabricated valves made from polymer diaphragmsare also discussed.

Martynova et al. (1997) report fabrication of plastic(poly(methylmethacrylate)(PMMA)) microfluid channels by imprintingmethods for use in electrophoretic and chromatographic applications.Anal. Chem., 69:4783-4789. Mechanical pumping is referenced, as are thefabrication techniques of casting, molding, laser ablating and machiningplastic. Bubble entrapment is noted as a problem that can be solved byheating.

Roberts et al. (1997) report micro channel construction using UV lasermachined polymer substrates (e.g., polystyrene, polycarbonate, celluloseacetate, and poly(ethylene terephthalate) (PET) for the development ofmicrodiagnostic systems. Anal. Chem., 69:2035-2042. The article alsodiscusses laminate sealing. Pump and valving are not addressed.

Burns et al. (1998) report an integrated nanoliter DNA analysis devicemade of a glass and silicone substrate and containing microfabricatedchannels, heaters, temperature sensors, and fluorescence detectors.Science, vol. 282, pp. 484-487. The device is made usingphotolithography and is reportedly capable of measuring, mixing,amplifying and digesting DNA.

Kopp et al. (1998) report continuous-flow PCR on a glass microchip.Science, vol. 280, pp. 1046-1048.

Waters et al. (1998) report a microchip device for cell lysis, multiplexPCR amplification, and electrophoretic sizing and fluorescence-baseddetection. Anal. Chem., 70:158-162. Microchannels are polymer-etched and50 um wide by 10.4 um deep. Valving, pumps, and the identity of thepolymer substrate are not addressed.

Duffy et al. (1999) report microfabricated centrifugal microfluidicsystems having microscopic channels formed in a plastic disk by castingmolded PDMS or machining pmethylmethylacrylate. Anal. Chem.71:4669-4678. The channels are reported to have diameters of 5 um-0.5 mmand depths of 16 um-3 mm.

Anderson, J. et al. (2000) report fabrication of three-dimensionalmicrofluidic systems in PDMS using “membrane sandwiches”, in which thinmembranes having channel structures molded on each face are fixed underpressure between two thicker, flat slabs. Anal. Chem. 72, pp 3158-3164.

Anderson, R. et al. (2000) report a miniaturized integratedpolycarbonate device (“disposable cartridge”) the size of a credit cardfor automated multistep genetic assays. Nucl. Acid. Res., Vol. 28, No.12, pp. i-vi. The device employs laminate valves made out of a 0.01 mmthick mylar held in place by ultrasonic welding or adhesives. Fluids aremoved therethrough using a pneumatic diaphragm valve and vacuum. Poroushydrophobic membranes are reported that allow the passage of gas but notliquids.

Barker et al. (2000) report polystyrene, PDMS, polycarbonate andpolyethylene terephthalate glycol (PETG) plastic microfluidic deviceshaving surfaces modified with polyelectrolyte multilayers (PEMs). Anal.Chem. 72: 4899-4903.

Beebe et al. (2000) report a PDMS microfluidics platform that combinesliquid-phase photopolymerization cartridges using lithography, channels,pH-actuated hydrogel valving, and sensors. PNAS, vol. 95, no. 25, pp.13488-13493.

Liu et al. (2000) report chaotic advection passive mixing in athree-dimensional serpentine microchannel having a C-shaped repeatingunit. J. Micro-electromech. Sys., Vol. 9 No. 2, pp. 190-197. The deviceis fabricated in a silicon wafer using a double-sided KOH we-etching.Discussed are active mixing techniques versus passive techniques, therelative sophistication and difficulties presented by the former, andthe need for at least one such mechanism when small dimension (tens ofmicrometers) channels are employed. Also discussed is the fluid dynamicsprinciple of Reynolds numbers, R_(e),=Q/A (flow rate overcross-sectional area)×D_(h)/v (hydraulic diameter of channel overkinetic viscosity of fluid. The repeating C-units emanate away from theinlet and toward a distinct outlet.

Oleschuck et al. (2000) report trapping of bead-based reagents withinmicrofluidic systems and on-chip solid-phase extraction andelectrochromatography, coupled with electrofluorescence detection. Anal.Chem., 72:585-590. The system is made of etched glass and featurescontinuous, valveless flow.

Unger et al. (2000) report on monolithic microfabricated valves andpumps for multilayer soft lithography. Science 288; 113-116. Softlithography is described as an alternative to silicon-basedmicromachining and uses replica molding of nontraditional elastomericmaterials to fabricate stamps and microfluidic channels, with advantagesafforded in terms of rapid prototyping, ease of fabrication andbiocompatibility. Systems containing on-off valves, switching valves,and pumps made entirely out of elastomer are described. Those systemsinclude microlectromechanical structures (“MEMS”) that are either bulkor surface micromachined from silica or other semiconductor-typematerials (e.g., polysilicon, metals, silicon nitride, silicon dioxide,etc.), with the latter sequentially applied and patterned in 3Dstructures, or else replication molding-based by patterned curing ofelastomeric material (“soft lithography”). The elastomer used is atwo-component addition-cured silicon rubber fused by hermetic sealingand irreversible bonding. Up to seven (7) independent layers arecombined into one using this technique. Each of the layers and resultingdevice is monolithic (i.e., all made from the same material). The valvesdescribed are crossed-channel in architecture, 100 um wide by 10 umhigh, mediated by polymer membrane typically 30 um in thickness, andsealed with a glass bottom layer. The flexibility and durability of thelayers permits the repeated opening and closing of valves upon pneumaticactuation without appreciable fatigue. Tubular flow channels are urgedas opposed to rectangular or other shapes, and there is also discussionof the problem of electrolytic bubble formation and avoidance thereof. Aperistaltic pump consisting of three valves arranged in a single channelis also reported. The figures also show unidirectional flow in which theinlets are remote from the “waste” outlet points.

Xu et al. (2000) report a room-temperature imprinting method formicrochannel fabrication in PMMA. Anal. Chem., 72:1930-1933. PDMS filmis used to seal the channels, which are imprinted from a micromachinedsilicone template. Pumping and valving are not per se addressed.

Chabinyc et al. (2001) report an integrated fluorescence detectionsystem in combination with disposable PDMS microfluidic implements.Anal. Chem., 73:44914498.

Gioradano et al. (2001) report use of the polymerase chain reaction(PCR) in polyimide microchips using 1.7 ul volumes and IR-mediatedthermocycling. Anal. Biochem., 291:124-132.

Ismagilov et al. (2001) report multi-phase laminar fluid flow and“switching” through a three-dimensional elastomeric microstructureformed by two microfluidic channels, fabricated in layers that contactone another face-to-face (typically at a 90 angle), with the fluid flowsin tangential contact. Anal. Chem. 73:4682-4687. There is no discussionof valves or valving per se, pressure is administered by syringe,polydimethylsiloxane (PDMS) membranes of 4-5 mm in dimension are used inconstruction, and channels of ˜25-200 μm operative height, 100-400 μmoperative width, and 2-4 cm operative length are used. Further, theinlet and outlet ports are remote to another, the adhesion of theindividual layers is accomplished by oxidizing the mating surfaces in anair plasma system for approximately 1 minute, and a glass cover slip isalso used.

Kamholz and Yager (2001) theoretically analyze molecular diffusion inpressure-driven laminar flow in microfluidic channels. Biophys. J.,80:155-160. The authors conclude there is reduced diffusivity inmicrofluidic systems, including, e.g., systems employing self-assemblingmonolayers (SAMS).

Lachner et al. (2001) report the advantages of planar microchipcapillary electrophoresis in conjunction with electrochemistry,including miniaturization potential while preserving sensitivity.Electrophoresis 22:2526-2536. Emphasis is on the selective grounding ofa detection reservoir relative to a “separation channel” into whichsample is first introduced. The system also features sample waste andbuffer reservoirs, as well as a high-voltage source to effectseparation. By definition, the system depends on electric fieldestablishment for sample migration and there is no circulation orrecirculation of liquid sample. These systems feature glass or plasticchips, with the latter fashioned from laser ablation or injectionmolding techniques, and a variety of electrode surfaces, includingcarbon, platinum, palladium, copper and gold. Applications discussedinclude those for separation and detection of catecholes, amino acids,peptides, carbohydrates, nitroaromatics, PCR products, organophosphatesand hydrazines. As expected for electrophoretic applications, separationof PCR products is coordinated with restriction enzyme digestion.

Whitesides et al. (2001) review soft lithograpy techniques and theimplications for microfabrication and biochip patterning andconfiguration. Annu. Rev. Biomed., 3:335-73. Soft lithography, asopposed to photolithography, is based on printing and molding usingelastomeric stamps with patterns of interest in bas-relief. PDMS is asubstrate of choice that is patterned with self-assembling monolayers(SAMs) and microcontact printing (uCP). Membrane-stacking is noted as amethod of synthesizing and configuring 3-dimensional microfluidicstructures. Pumps (including pneumatic) and soft PDMS membrane flapvalving is also briefly noted.

Yuen et al. (2001) report a microchip module of blood sample preparationand nucleic acid amplification reactions. Genome Res., 11:405-412. Themodule is a computer numerical control-machined Plexiglas microchip. Asyringe pump is used in tandem with valving.

Auroux et al. (2002) review micro analysis systems for the period 1997to 2002. Anal. Chem. 74: 2637-2652.

Beebe et al. (2002) review microfluidics in general and the fabricationof valves, mixers and pumps for the same as of 2002. Annu. Rev. Biomed.Eng. 4:261-86. Micromatching, soft lithography, embossing, in situconstruction, injection molding and laser ablation are discussed, aswell as the advantages and disadvantages attendant thereto.

Jeon et al. (2002). Report design and fabrication of integrated passivevalves and pumps for flexible polymer 3-dimensional microfluidicsystems. Biomed Microdevices 4:117-121.

Johnson et al. (2002) report rapid microfluidic mixing in preformedT-microchannel imprinted in a hot-imprinted polycarbonate siliconstamped substrate and modified with a pulsed UV excimer laser to createslanted wells at the junction. Anal. Chem. 74:45-51. PETG “lids” weresealed to the PC by heat-bonding.

Stroock et al. (2002) report chaotic mixers for microchannels. Science295: 647-651. The difference between laminar and turbulent flow isdiscussed in terms of efficient mixing, with the former (characteristicof systems having channels of dimension ˜100 μm or less) described asless efficient and characteristic of microfluidic systems in general.Stroock et al.'s solution is to employ textured relief structuresdeposited by planar lithographic techniques inside PDMS microfluidicchannels in order to impart differential resistance across variedtopographic surfaces, thereby improving passive mixing in the process.

Klank et al. (2002) report CO₂ laser micromachining and back-endprocessing for rapid production of PMMA-based microfluidic systems. LabChip, 2:242-246.

McDonald and Whitesides (2002) report poly(dimethylsiloxane) (PDMS) as auseful material for fabricating microfluidic devices. Accounts ofChemical Res., vol. 35, no. 7, pp. 491499. Silicone adhesive tapes arenoted for their ability to reversibly effect water-tight binding betweendifferent PDMS components. 3-D “membrane sandwich” fabrication bystacking multiple layers is also discussed, as is the ability toconfigure the devices with chambers that fit pipette tips.

Pugmire et al. (2002) report surface characterization of laser-ablatedpolymers used for microfluidics. Anal. Chem., 74:871-878. Electroosmoticflow comparisons are made between PMMA, PETG, PVC and PC after ablationunder different gaseous conditions. Pumping and valving are notaddressed.

Qi et al. (2002) report high-aspect-ration microstructures (HARMS) inmicrofluidic devices fabricated from PMMA using hot-embossing withintegrated sampling capillary and fiber optics for fluorescencedetection. Lab Chip, 2:88-95. Aspect ratio is described as the ratio offeature height to lateral dimension. Pumping and valving are notaddressed.

Reyes et al. (2002) briefly review the historical evolution of micrototal analysis systems (“uTAS”; synonymous with “lab on a chip”) theoryand technology, including microfabrication, bonding, surfacemodification, design, interfaces and connections, microvalves and flowcontrol, and micropumps. Anal. Chem. 74:2623-2636. Constructionmaterials noted include, e.g., PDMS, PMMA, PC,poly(ethyleneterephthalate) (PET), andpoly(tetrafluoroethylene)(Teflon®). Valving and pumping are generallydiscussed on page 2631 et seq. Passive membraneous check valves arenoted, as well as flap, lever and duckbill varieties.

Wang, J. (2002) reports on electrochemical detection in microscaleanalytical systems and how such systems offer possible advantages by wayof miniaturization, portability and, more tenuously, disposability.Talanta 56:223-231. Emphasis is again on electrophoretic separations andsample reservoir to waste reservoir directionality and flow, with thereservoirs made of PDMS/glass. Included is discussion of capillaryelectrophoretic (CE) systems, micromachining and ablation techniques, aswell as discussion of different electrode types/compositions and formsof electronic detection, e.g., fixed-potential/current monitoring(amperometric) and voltammetry. Detection of nucleic acids is discussedusing intercalating iron-phenanthroline redox markers.

Breadmore et al. (2003) report microchip-based purification of DNA frombiological samples. Anal. Chem. 75:1880-1886.

Fiorini et al. (2003) report fabrication of thermoset polyestermicrofluidic devices and embossing masters using rapid prototypedpolydimethylsiloxane (PDMS) molds. Lab Chip, 3:158-163.

Glasgow and Aubry (2003) report enhanced microfluidic mixing using timepulsing. Lab Chip, 3:114-120. Mixing is accomplished by time varying andpulsing fluid flow using, e.g., variable channel dimensions. Bubbleentrapment is stated as a problem to be avoided. The substrate used isnot identified and there is no discussion of venting or valving.

Jensen et al. (2003) report microstructure fabrication in poly(methylmethylacrylate) (PMMA) with a CO₂ laser system, including rasterscanning to produce cavities 50 um wide and 200 um deep. Lab Chip,3:302-307.

Koh et al. (2003) report integrated PCR, valving and electrophoresis ina plastic device for bacterial detection. Anal. Chem. 75:45914598. Thedevice is made from cyclic polyolefin having graphite ink electrodes andphotopatterned gel domains that function as passive valves. Detection isoptical and accomplished using laser-induced fluorescence of aninterchalating dye. Volumes used were 29-84 nL.

Kricka and Wilding (2003) review microchip PCR systems bearingserpentine channels and fabricated from molded PDMS, micromachinedpolycarbonate or assembled layers of ceramic tape, held together, e.g.,by use of adhesives. Anal. Bioanal. Chem. 377:820-825. A host ofpassivation agents are also discussed that avoid adverse surfaceinteractions, including, e.g., silicon oxide, PDMS, polypropylene, BSA,and polyvinylpyrrolidone.

Landers (2003) reports inter alia on the potential for performing singlenucleotide polymorphism (SNP) diagnostics on electrophoretic microchips,preferably using optical detection on capillary based systems. Anal.Chem., 75:2919-2927. See, e.g., pp. 2922.

Liu et al. (2003) report sophisticated microfluidic PCR systems devisedof multilayer elastomeric PDMS formed using photolithography and activepumping and valving schemes. Anal. Chem. 75:4718-4723.

Wang et al. (2003) report low-density microarrays assembled inmicrofluidic chips fabricated from hot-embossed PMMA for the detectionof low-abundant DNA mutations. Anal. Chem., 75:1130-1140. Appropriateligand linking chemistry is also addressed.

Buch et al. (2004) report DNA mutation detection in a modularpolycarbonate microfluidic network using temperature gradient gelelectrophoresis. Anal. Chem. 76:874-881. One module is embossed withmicrochannels and the other contains a tapered microheaterlithographically patterned along with an array of temperature sensors.

Gustafsson et al. (2004) report integrated peptide sample preparationand MALDI Mass Spectometry on a Microfluidic Compact Disk, in whichsample fluid is pushed using centripetal force. Anal. Chem. 76:345-350.

Hashimoto et al. (2004) report rapid PCR in a continuous flow embossedpolycarbonate device. Lab Chip, 4:638-645. Microchannel dimensions usedwere 6 cm(L)×50 um(W)×150 um(H).

Howell et al. (2004) report on fluid dynamics principles and the designand evalution of a Dean vortex-based micromixer on a machined PMMA chip.Lab chip, 4:663-669. A double-sided adhesive tape is used to fix themachined chip to a glass slide and bubble avoidance is urged.

Lagally et al. (2004) report an integrated portable genetic analysismicrosystem for pathogen/infectious disease detection using PCR,electrophoresis and laser-excited fluorescence detection. Anal. Chem.,76:3162-3170. The system is said to be of etched glass wafer design andcontain active solenoid PDMS “membrane valves”, with one particularconfiguration possessing three such valves in series to collectivelyform a “diaphragm” pump. Sample volumes are 200 nL.

Lai et al. (2004) report a resin-gas injection packaging technique forbonding and surface modification of polymer-based microfluidic platformssuch as glass, silicon, polyethylene, polystyrene poly(methylmethacrylate) (PMMA), polyamide, and polycarbonate. Anal. Chem.,76:1175-1183. Also noted are adhesive layer techniques and the problemof bubble accumulation/obstruction and the suggestion to use a vacuum tominimize such.

Laser and Santiago (2004) review micropump structures in J. Micromech.Microeng., 14:R35-R64. The detrimental problem of bubbles inmicrofluidic systems is noted repeatedly throughout, as is the generaldearth of effective pumping systems in microfluidics systems. Despitethis, reciprocating pneumatically-driven diaphragm pumps flanked bypassive check valves are discussed in the context of multilayerconstructions, see, e.g., FIG. 1 and § 2.1, although diaphragms made outof soft polymer membranes are said to be a “concern” because ofstability. Etching, micromachining and photolithography are alsodiscussed as means of creating device channels and chambers.

Noerholm et al. (2004) report a disposable polycarbonate microfluid chipfor online monitoring of microarray hybrizations. Lab Chip, 4:28-37. Thechip is 25×76×1.1 mm in dimension and manufactured by micro injectionmolding. The chip is said to contain an inlet, a 10 ul hybridizationchamber, a waster chamber and a vent to allow air to escape when sampleis injected. Its utility is demonstrated using hybridization buffer,wash buffers, fluorescence-based detection and a computer controlledsyringe pump. The system would appear to be capillary-action mediated,continued flow, non-recirculating and valveless. Use of plastic polymersis said to endow advantages by way of milling, laser ablation, hotembossing and injection molding. The use of adhesive tape in fashioningmicrostructures is also noted. The problem of bubble development is alsonoted but the vent used is located remote to the inlet and proximal to awaste chamber.

Schonfeld et al. (2004) report an optimized split-and-recombine (SAR)micro-mixer formed from milled PMMA and featuring active, uniform“chaotic” mixing. Lab Chip, 4:65-69.

Vilkner et al. (2004) review various micro total analysis systems(uTAS), including microfabrication, bonding techniques, microvalves andflow control, and micropumps. Anal. Chem., 76:3373-3386. Their reviewbuilds on that of Reyes (2002) and, in addition to discussing PDMS,PMMA, PC, poly(ethyleneterephthalate) (PET), andpoly(tetrafluoroethylene) (Teflon®) as construction materials, andgeneral valving, further includes discussion, e.g., of thermoresponsivehydrogel plugs and valving.

Yaralioglu et al. (2004) report ultrasonic mixing in PDMS microfluidicchannels using integrated piezoelectric transducers. Anal. Chem.,76:3694-3698.

Fiorini and Chiu (2005) review disposable microfluidic devicefabrication, function and application. BioTechniques vol. 38, no. 3, pp.429-446. Methods of fabrication include replica and injection molding,embossing, and laser ablation. Fluid pumping and valving is alsodescribed, as is mixing and analyte separation and detection. Deformablemembrane pressure pumps and valves are particularly discussed at pp.434-5, as is the concept of pulsatile flow. Strategies for mixinginclude use of 3-dimensional serpentine channels. P. 435. Multilayerfabrication with plastics is also mentioned, as are electrochemicaldetection schemes and advantages attendant thereto, and nucleic acids asdetectable analyte. pp. 438-9.

Howell et al. (2005) report a microfluidic mixer with grooves placed onthe top and bottom of milled PMDA channels. Lab Chip, 5:524-530.

Klapperich et a. (2005) report hot-embossed fabrication of a cyclicpolyolefin microfluidic device for on-chip isolation of nucleic acidsonto silicon particles embedded in the device, followed by elution.Proc. ICMM2005, 3^(rd) Int. Conf. on Microchannels and Minichannels.Toronto, CANADA.

Lee et al. (2005) report development of a passive 3-dimensional PDMSmicromixer based on repeated fluid twisting and flattening of thechannels, and its application to DNA purification. Anal. Bioanal. Chem.,383:776-782. Multi-layer stacking and multi-step photolithography arenoted as device fabrication techniques. The system has discreet inletsand outlets that are remote relative to one another.

Roper et al. (2005) report advances in polymerase chain reaction (PCR)methodology on polycyclic olefin microfluidic chips using hydraulicvalves and pneumatic pumps. Anal. Chem., 77:3887-3894. Reported reactionvolumes are approximately 30 nl.

Skelley et al. (2005) report development and evaluation of asophisticated capillary electrophoresis microdevice made of glass wafersand PDMS membranes for amino acid biomarker detection and analysis useon Mars. PNAS, vol. 102, no. 4, pp. 1041-1046. The device is vacuumdriven and said to possess 34 individual membrane valves and 8 pumps.The wafers are 10 cm in diameter with 20 um deep×70 um wide×21.4 cm longchannels.

Wang et al. (2005) report label-free detection of small-molecule-proteininteractions using nanowire nanosensors (silicone; SiNW) and fieldeffect transistors (FETs) on a surface plasma resonance (SPR)-like chip.PNAS, vol. 102, no. 9, pp. 3208-3212.

Whitesides et al. (2005) report a technique for storing and delivering asequence of reagents to a microfluidic device. Abstract, Anal. Chem.,77(1):64-71. The technique makes use of cartridges of tubing filled bysequentially injecting plugs of reagents separated by air spacers.

Liu et al. (2006) report integrated microfluidic biochips for DNAmicroarray analysis by fluorescence imaging that containelectromechanical pumps, low-cost check valves, fluid channels andreagent storage containers. Expert Rev. Mol. Diagn., 6(2):253-261(Abstract).

Soper et al. (2006) forecast point-of-care (POC) biosensor systems forcancer diagnostics/prognostics. Biosensors and Bioelectronics,21:1932-1942. The article only generally speaks to the future of thefield and the need for mass-production, low cost fabrication andspecialized valving and pumping systems. Techniques contemplated forconstruction of such devices include injection molding, nanoprintlithography and hot-embossing.

U.S. Pat. Nos. 7,101,509 and 6,368,87,1 assigned to Cepheid share acommon specification and collectively report and claim temperaturecontrolled devices and methods for the manipulation of materials in afluid sample using a plurality of microstructures bearing insulatorfilms (selected from silicon dioxide, silicon carbide, silicon nitride,and electrically insulating polymers). The devices employ integratedloading chambers, reaction vessels, and aspirators in connection withthe insulator-film bearing structures. Application of a voltage to thestructures induces the desired electrophoretic separation andattraction, followed by washing and elution steps. U.S. Pat. Nos.6,893,879, 6,664,104 and 6,403,037 assigned to Cepheid report and claimsimilar analyte flow, capture and elution techniques and devices.

U.S. Pat. No. 6,818,185 reports and claims a cartridge for conducting achemical reaction that consists of a body having at least first andsecond channels formed therein, a reaction vessel extending from thebody, a reaction chamber, an inlet port connected to the reactionchamber via an inlet channel, and an outlet port connected to thereaction chamber via an outlet channel. The inlet port of the vessel isconnected to the first channel in the body, and the outlet port of thevessel is connected to the second channel in the body. The walls of thereaction chamber contain polymeric films, and vents for exhausting gasfrom the second channel are also described. The system also employsdifferential pressure sources for forcing sample through the system,which can further include thermal surfaces, heating elements, mixing andlysing chambers, and optically transmissive walls.

U.S. Pat. No. 6,374,684 reports a fluid control and processing systemhaving a plurality of chambers and a valve body that includes a fluidsample processing region coupled with a fluid displacement region, thefluid displacement region depressurizable to draw fluid into the fluiddisplacement region and pressurizable to expel fluid from the fluiddisplacement region.

U.S. Pat. Nos. 6,830,936, 6,197,595, 6,043,080, 5,922,591, and5,856,174, each entitled “Integrated nucleic acid diagnostic device” andassigned to Affymetrix, describe and/or claim diaphragm or controllablevalve actuated miniature fluid flow systems for measuring and processingfluid samples. The systems described make use of a plurality ofdifferent chambers and channels, as well as inlet and vent ports. U.S.Pat. Nos. 6,733,977 and 6,168,948 are similar to these in content.

U.S. Pat. Nos. 7,223,363 (entitled “Method and System for MicrofluidicInterfacing to Arrays”) and 7,235,400 (entitled “Laminated MicroarrayInterface Device”), each assigned to BioMicro Systems Inc., reportpump-driven multi-laminate microfluidics systems having a gasket thatdefines the walls of a reaction chamber (e.g., serpentine), integratedpassive valving, diaphragm and multiple bladder use to promote mixing,the possibility for sample (re)circulation and bubble elimination usingan external pumping scheme, the relative positioning of multiple suchdevices such that there is a pitch of 9 mm between devices for ease ofloading by a multipipetteman, and the merit of using constructionmaterials that permit visualization/optical assessment of the system.

U.S. Pat. Nos. 5,063,081 (“Method of manufacturing a plurality ofuniform microfabricated sensing devices having an immobilized ligandreceptor”), 5,096,669 (“Disposable sensing device for real time fluidanalysis”) and 5,124,661 (“Reusable test unit for simulatingelectrochemical sensor signals for quality assurance of portable bloodanalyzer instruments”) to I-STAT Corporation discuss inter alia use of adisposable cartridge system that makes use of an internal bladder tomanipulate liquid sample.

Micronics, Inc. also holds numerous patents in the field ofmicrofluidics including, e.g., U.S. Pat. Nos. 7,223,371 (“Microfluidicchannel network device”), 6,743,399 (“Pumpless microfluidics”),6,742,661 (“Well-plate microfluidics”), 6,581,899 (“Valve for use inmicrofluidic structures”), 6,557,427 (“Capillaries for fluid movementwithin microfluidic channels”) and 6,488,896 (“Microfluidic analysiscartridge”).

Numerous patents and papers published by Paul Yager are also germane tothe topic of microfluidics and include, e.g., U.S. Pat. Nos. 5,716,852and 5,972,710 (“Microfabricated diffusion-based chemical sensor”),6,007,775 (“Multiple analyte diffusion-based chemical sensor”),6,039,897 (“Multiple patterned structures on a single substratefabricated by elastomeric micro-molding techniques”), 6,110,354 and6,790,341 (“Microband electrode arrays”), 6,159,739 (“Device and methodfor 3-dimensional alignment of particles in microfabricated flowchannels”), 6,454,945 (“Microfabricated devices and methods”), Sensorsin Biomaterials Science: An Introductory Text, Ratner, B. D. andHoffman, A. S., Eds. Academic Press, Inc., Orlando, (1996), Low Reynoldsnumber micro-fluidic devices, Proceedings Hilton Head MEMS conference,Solid-State Sensor and Actuator Workshop, 105-108, (1996), Biotechnologyat low Reynolds numbers, Biophysical Journal. 71 (6), 3430-3441, (1996),Integration of microelectrodes with etched microchannels for in-streamelectrochemical analysis, Micro Total Analysis Systems, 105-108 (1998),Design of microfluidic sample preconditioning systems for detection ofbiological agents in environmental samples, SPIE Proceedings, 3515,252-259 (1998), Whole blood diagnostics in standard gravity andmicrogravity by use of microfluidic structures (T-sensors), MikrochimicaActa, 131, 75-83 (1999), A novel microfluidic mixer based on successivelamination, Micro Total Analysis Systems, Mesa Monographs, 495-498(2003), On the importance of quality control in microfluidic devicemanufacturing, Micro Total Analysis Systems, Mesa Monographs, 1069-1072(2003), Lab-on-a-chip and fluorescence sensing on the microscale,Fluorescence Sensors and Biosensors, R. B. Thompson, ed., ISBN0-8247-2737-1, CRC Press, Boca Raton, Fla., c. 400 pp (2005), Rapid,parallel-throughput, multiple analyte immunoassays with on-boardcontrols on an inexpensive, disposable microfluidic device, Micro TotalAnalysis Systems, Vol. 2, Transducer Research Foundation, Pubs.,1000-1002 (2005), Recirculating flow accelerates DNA microarrayhybridization in a microfluidic device, Lab on a Chip, in press.

Microfluidic systems and function is also addressed in patents andpublications by Stanford's Stephen Quake, including U.S. Pat. Nos.7,232,109 (“Electrostatic valves for microfluidic devices”), 7,216,671,7,169,314, 7,144,616, 7,040,338, 6,929,030, 6,899,137, and 6,408,878(“Microfabricated elastomeric valve and pump systems”), 7,143,785(“Microfluidic large scale integration”), 6,960,437 (“Nucleic acidMicrofabricated elastomeric valve and pump systems, 6,793,753 (“Methodof making a microfabricated elastomeric valve”), 6,767,706 (“Integratedactive flux microfluidic devices and methods”), and “A nanoliter-scalenucleic acid processor with parallel architecture,” Nat. Biotechnol, 22:4: 435-9 (2004), “Solving the “world-to-chip” interface problem with amicrofluidic matrix.” Anal. Chem., 75: 18: 4718-23 (2003),“Microfluidics in structural biology: smaller, faster em leader better.”Curr. Opin. Struct. Biol. 13: 5: 538-44 (2003), “Integrated nanolitersystems,” Nat. Biotechnol., 21: 10: 1179-83 (2003), “Microfabricatedfountain pens for high-density DNA arrays,” Genome Res., 13: 10: 2348-52(2003), “Microfluidic memory and control devices,” Science, 300: 5621:955-8 (2003), “Microfluidic large-scale integration,”Science, 298: 5593:580-4 (2002), “A nanoliter rotary device for polymerase chain reaction,”Electrophoresis, 23:10:1531-6 (2002), “Dynamic pattern formation in avesicle-generating microfluidic device.” Phys. Rev. Lett. 86: 18: 4163-6(2001), “Monolithic microfabricated valves and pumps by multilayer softlithography.” Science, 288: 5463: 113-6 (2000); “From micro- tonanofabrication with soft materials,” Science, 290: 5496: 1536-40(2000), and “A microfabricated device for sizing and sorting DNAmolecules.” Proc. Natl. Acad. Sci. USA 96: 1: 11-3 (1999).

In addition to the foregoing work of others, commonly-owned U.S. Pat.Nos. 7,172,897, 6,960,467, 6,875,619, 6,833,267, 6,761,816, 6,642,046,6,592,696, 6,572,830, 6,544,734, 6,432,723, and 6,361,958 also speak tomicrofluidics and microfluidics operations, including integration ofindividual electronic components and positionment into detectiondevices, including electrochemical detection devices.

As will be become apparent, the configuration and function of the abovethird party devices is different from aspects and embodiments of theinventions described herein in one or more of construction, valving,mixing, diaphragm positionment and function, bubble elimination, pumpinterfacing and recirculation design. These differences give rise toreal advantages and prospects for the inventions described herein.

BRIEF SUMMARY OF THE INVENTION

Accordingly, in one aspect the invention provides fluidics devicescomprising at least one membranous diaphragm having a first and secondside. The first side is for coupling to a pump, and the second side isfor fluidic coupling to a flow channel (which can include a detectionchamber as a flow channel) and a plurality of check valves. The checkvalves each comprise a sealing surface comprising a valve seat thatallows for regulated flow of fluid through the flow channel and aflexible sealing structure for contacting the valve seat and occludingfluid flow therethrough when in a first position, and for promotingfluid flow therethrough when in a second position. Generally, in thisaspect, the diaphragm and the flexible sealing structure are integral toone or more layers of a multilayer assembly (e.g. a multilaminatestructure). The plurality of check valves alternate opening and closingin coordinated reciprocal fashion according to alternating positive andnegative forces exerted on the diaphragm. In an additional aspect, thedevice comprises a body comprising a channel (which can be the detectionchamber) within for transporting a fluid sample, the channel comprisinga situs where an operation is performed on one or more components in thefluid sample. The body also comprises an inlet port for receiving thefluid sample, the inlet port in fluid communication with the channel andalso in communication with an enclosed gaseous environment. The inletport can be configured to receive the fluid sample from a pipette tip,including micropipette tips. The body also comprises an outlet port influid communication with the inlet port such that upon operation of thedevice the fluid sample is recirculated through the channel to releasegaseous bubbles into the enclosed gaseous environment whilesimultaneously allowing for facilitated diffusion and performance of theoperation on the one or more components in said fluid sample.

In a further aspect, the fluidics devices process no more than about 1.5mL of fluid at a time, with some aspects processing no more than about150 ul of fluid at a time. In some aspects, the sample capacity of thedevice is from about 50 ul to about 200 ul. The channels can comprise across-sectional dimension comprising a greater width than height. Insome aspects, the channels have a cross-sectional dimension ranging fromabout 0.030″ to about 0.060″ in width and from about 0.006″ to about0.014″ in height.

In an additional aspect, the fluidics devices are made at least in partfrom the stacking of multiple individual polymeric laminate sheets,optionally held together by pressure sensitive adhesive sheets. In someaspects, the devices are constructed from alternating laminate andadhesive layers having individual thicknesses in the range of from about0.0005″ to about 0.030″. In some aspects, the individual laminate layersare thicker than the individual adhesive layers. In some aspects, theindividual laminate layers are from about 0.0005″ to about 0.010″ thickand said individual adhesive layers are from about 0.001″ to about0.003″ thick.

In a further aspect, one or more of the channels of the fluidics devicesare formed by grooves, cuts or recesses in one or more of theseindividual plastic laminate sheets, which can be produced by adie-stamp, laser, chemical etching, or molding.

In an additional aspect, the channels that serve as detection chambersfurther comprise at least one working electrode in the flow channel,optionally comprising capture binding ligands and SAMs, and thus isoptionally a diagnostics device. Thus, aspects of the invention providefor immobilized biological binding partners in the flow/detectionchannel. These biological binding partners can be immobilized using aself assembling monolayer that is affixed to a surface, optionally anelectrode. The biological binding partners can be polynucleotides orproteins (including peptides and antibodies).

In a further aspect, the fluidics devices of the invention furtheroptionally comprise one or more auxiliary electrodes and/or connectorsfor interface with a detection device. In some aspects, the connectorsare configured in a two-dimensional grid of contact points. In someaspects, the connectors may be ZIF connectors and/or side or edgeconnectors. The fluidics devices can also optional comprise an EEPROM,and/or an internal unused space designed for integration of one or morefuture functionalities.

In an additional aspect, the fluidics devices of the invention cancomprise a transparent or translucent plastic (e.g. an optical window)that permits visualization of circulation and recirculation of the fluidsample within.

In a further aspect, the fluidics devices can comprise one or morevalves in fluid communication with the channel for regulating the flowof the fluid sample therethrough. The valves can be check valves,passive valve and bridge valves. In some aspects, or more of of thevalve seats protrude from a planar structure to promote sealing uponengagement with a corresponding sealing structure. The magnitude of theprotuberances relative to the base surface is independently selectedfrom about 0.001″ to about 0.005″. Optionally, one of more of the valveseats are part of a hollow boss having a plurality of ports adjacent andfluidly coupled to one another in a nonlinear configuration, optionallya “u-structure”, and routing fluid from one fluidics plane in the deviceto one or more other fluidics planes in the device.

In an additional aspect, the fluidics devices can further comprise ameans for pumping (e.g. a pump) the fluid sample through the channel.Thus, the devices can comprise a plurality of bridge or check valvesactuated by a pneumatic, electromagnetic, or hydraulic pump and adiaphragm. The pneumatic pump can be electrically driven.

In an additional aspect, the devices can further comprise caps forsealing the device following the addition of a sample, and optionally afilter, adsorbent and/or absorbent for reducing or eliminating solutesor analytes in the fluid sample.

In a further aspect, the fluidics devices of the invention furthercomprise a detector that detects binding events between binding partnersand complementary binding partners in a fluid sample, which can be acolorimetric detector or an electronic detector that detects electronicproperties of the binding events. In some cases, electrochemiluminescentdetection is not preferred. In some cases, fluorescent detection is notpreferred.

In an additional aspect, the fluidics devices of the invention aremicofluidics devices, filtration devices, or purification or abstractiondevices.

In some aspects, particularly when bubble removal is desired, thefluidics devices receives the fluid sample through the inlet port in anupright position of from 15°-90° relative to horizontal and processesthe fluid sample in a 15°-90° position relative to horizontal, andwherein the receiving upright position and the processing uprightposition are not necessarily the same.

In a further aspect, the invention provides racks for carrying aplurality of fluidics devices of the invention, wherein the rack isdesigned to position said devices relative to one another having a pitchof about 9 mm between successive sample reservoirs of said devices.

In an additional aspect, the invention provides devices that arediaphragm-mediated two-stroke circulation and recirculation devicesmediated by passive valving.

In a further aspect, the invention provides diagnostic kits comprisingthe devices of the invention, and optionally reagents.

In an additional aspect, the invention provides methods of determiningthe presence, absence and/or amount of analyte in a sample, or analytebinding. The methods comprise providing a fluidics device as outlinedherein, adding a sample (usually liquid) suspected of containing one ormore of the analytes, circulating and recirculating the sample acrossthe array; and detecting binding of the analytes to the ligands.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a perspective view of a fully assembled fluidics device(cartridge) according to one embodiment of the invention, showingintegral handle and cover, integral fill reservoir, pump interface, andcap. Also shown is one of two detents to help align cartridge uponinsertion into a cartridge module and provide tactile/audible feedback.FIG. 1B is an exploded view of the individual components within thecartridge.

FIG. 2 is another exploded view of FIG. 1 showing PCB (printed circuitboard), valve/diaphragm laminate assembly, plate, cover and cap, withvisible sample addition fill reservoir, serpentine channel, pumpinterface, bridge valves, and serpentine electrode array on PCB boardconfigured to interface with serpentine channel in laminate assembly tothereby create one or more fluid channels.

FIG. 3 is a perspective view of a partially assembled cartridgeembodiment (less cover) containing pump interface, sample fillchamber/reservoir (with cap), alignment holes, and EEPROM.

FIG. 4 is a bottom perspective view of a cartridge plate assemblyembodiment (less cover, cap and PCB), including alignment holes, voidfor receiving EEPROM, sample fill reservoir, sample inlet port, andsample outlet port. Also shown are two valve seats, one at the fillreservoir outlet and the second below the ports of the oval recess(above and to the right of diaphragm), a bubble stripping/fillreservoir, and diaphragm for interface with pump.

FIG. 5 is a perspective view of a partially assembled cartridge thatcontains the laminate assembly overlaying the PCB board and that bettershows the operational relation between bridge valves, diaphragm, andchannel.

FIG. 6 is a perspective view of a PCB board configured with reference,auxiliary and working electrodes in serpentine configuration, alignmentholes and EEPROM. Not visible are the traces and vias linking theindividual electrodes to a two-dimensional array of gold contact pointson the underside of the PCB board.

FIG. 7 shows the underside view of FIG. 6, including connector pads andrectangular area showing the thermal interface location for receipt of atemperature-controlled metal plate thereover. There are also alignmentfeatures (cut-outs or “cookie bites”) shown to facilitate properconnection to the connector. The connector pads are clamped into adetection device preferably using a zero-insertion force (ZIF)mechanism. In another embodiment, the connector pads may be concentratedin mass on a side facing (“edge” connectors).

FIG. 8 shows individual modular laminate components of the laminateassembly, each having features cooperative with and complementary toeach other when in operative use. The top piece is an adhesive layerwith voids for functional overlay over the middle laminate whichcontains bridge valve slits and orifices that cooperate with those onthe lower laminate/adhesive to form functional diaphragm, valves andchannels. The lower piece is a laminate covered on both sides with anadhesive layer, the bottom of which mates with the PCB board.

FIG. 9 is a top perspective view of the plate, which houses the samplefill reservoir, pump interface, void for receipt of an EEPROM and ovalrecess over which is sealed a jumper laminate. Also shown are raisedbosses that facilitate engagement of plate to cover.

FIG. 10 is a perspective view of a rack containing multiple cartridgesvertically aligned and stacked, with sample fill reservoirs facingupward for receipt of liquid samples from a liquid delivery system, suchas a multi-pipetteman.

FIG. 11 is a sectional view of a fully assembled cassette embodiment,showing cooperativity of the PCB board, laminate assembly, plate, andcover to afford fluidic operation. Shown are the channels, valving,diaphragm and pneumatic pump interface.

FIG. 12 is similar to FIG. 8, showing an exploded view of the individuallaminate and adhesive layers comprising the laminate assembly.

FIG. 13A shows an assembled cartridge module and FIGS. 13B and Crespectively show the bottom and top hemispheres of the module,including internal components and design. FIG. 13D shows the buckle beamassembly, with one bracket displaced showing buckle beams. FIG. 13E is aperspective view of the top of the module showing connection to pump.

FIG. 14 shows an embodiment of a detection device for housing multiplecartridges according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

Ease of use and cost considerations are driving systems into a paradigmwhere inexpensive, disposable consumables are used together with capitalequipment. This is especially true in analytic and diagnosticapplications where concern over possible contamination of one samplewith another leads consumers to prefer single-use devices wherefeasible. While the inventions discussed here apply more generally toany system configuration and to applications beyond diagnostics, thisdescription will focus on the use of a disposable device—acartridge—that manipulates sample-specific reagents in concert withseparate, reused instrumentation for diagnostic purposes.

Accordingly, in one embodiment the present invention is directed to afluidics device designed to analyze a plurality of target analytes. Inone embodiment the fluidics device of the present invention includes amembranous diaphragm, one side of which, in one embodiment, is coupledto a pump. The second side is coupled to a flow channel, and a pluralityof check valves for fluidic coupling. The valves include a sealingsurface that include a valve seat allowing for for regulated flow offluid through the flow channel, and a flexible sealing structure forcontacting the valve seat and occluding fluid flow through the sealingstructure when in a first position and for promoting fluid flow throughthe seal when in a second position. In one embodiment the diaphragm andflexible sealing structure are integral to one or more layers of amultilayer assembly. The plurality of check valves alternate opening anclosing in coordinated reciprocal fashion according to alternatingpositive and negative forces exerted ion the diaphragm.

In another embodiment the fluidics device includes a body. The bodyincludes a channel within it for transporting a fluid sample. Thechannel includes a situs where an operation is performed on one or morecomponents in the fluid sample. In addition the body includes an inletport for receiving the fluid sample. The inlet port is in fluidcommunication with the channel and also in communication with anenclosed gaseous environment. The body also includes an outlet port influid communication with the inlet port such that upon operation of thedevice the fluid sample is recirculated through the channel to releasegaseous bubbles into the enclosed gaseous environment whilesimultaneously allowing for facilitated diffusion and performance of theoperation on the one or more components in the fluid sample.

Thus, the present disclosure provides compositions and methods fordetecting the presence or absence of target analytes in samples. As willbe appreciated by those in the art, the sample solution may comprise anynumber of things, including, but not limited to, bodily fluids(including, but not limited to, blood, urine, serum, lymph, saliva, analand vaginal secretions, perspiration and semen, of virtually anyorganism, with mammalian samples being preferred and human samples beingparticularly preferred); environmental samples (including, but notlimited to, air, agricultural, water and soil samples); biologicalwarfare agent samples; research samples (i.e. in the case of nucleicacids, the sample may be the products of an amplification reaction,including both target and signal amplification as is generally describedin PCT/US99/01705, such as PCR amplification reaction); purifiedsamples, such as purified genomic DNA, RNA, proteins, etc.; raw samples(bacteria, virus, genomic DNA, etc.); as will be appreciated by those inthe art, virtually any experimental manipulation may have been done onthe sample.

The methods are directed to the detection of target analytes. By “targetanalyte” or “analyte” or grammatical equivalents herein is meant anymolecule or compound to be detected and that can bind to a bindingspecies, defined below. Suitable analytes include, but are not limitedto, small chemical molecules such as environmental or clinical chemicalor pollutant or biomolecule, including, but not limited to, pesticides,insecticides, toxins, therapeutic and abused drugs, hormones,antibiotics, antibodies, organic materials, etc. Suitable biomoleculesinclude, but are not limited to, proteins (including enzymes,immunoglobulins and glycoproteins), nucleic acids, lipids, lectins,carbohydrates, hormones, whole cells (including procaryotic (such aspathogenic bacteria) and eucaryotic cells, including mammalian tumorcells), viruses, spores, etc. Particularly preferred analytes areproteins including enzymes; drugs, cells; antibodies; antigens; cellularmembrane antigens and receptors (neural, hormonal, nutrient, and cellsurface receptors) or their ligands.

In one embodiment, the target analyte is a protein. As will beappreciated by those in the art, there are a large number of possibleproteinaceous target analytes that may be detected using the presentinvention. By “proteins” or grammatical equivalents herein is meantproteins, oligopeptides and peptides, derivatives and analogs, includingproteins containing non-naturally occurring amino acids and amino acidanalogs, and peptidomimetic structures. The side chains may be in eitherthe (R) or the (S) configuration. In one embodiment, the amino acids arein the (S) or L-configuration.

As discussed below, when the protein is used as a binding ligand, it maybe desirable to utilize protein analogs to retard degradation by samplecontaminants. Suitable protein target analytes include, but are notlimited to, (1) immunoglobulins, particularly IgEs, IgGs and IgMs, andparticularly therapeutically or diagnostically relevant antibodies,including but not limited to, for example, antibodies to human albumin,apolipoproteins (including apolipoprotein E), human chorionicgonadotropin, cortisol, a-fetoprotein, thyroxin, thyroid stimulatinghormone (TSH), antithrombin, antibodies to pharmaceuticals (includingantieptileptic drugs (phenytoin, primidone, carbariezepin, ethosuximide,valproic acid, and phenobarbitol), cardioactive drugs (digoxin,lidocaine, procainamide, and disopyramide), bronchodilators(theophylline), antibiotics (chloramphenicol, sulfonamides),antidepressants, immunosuppresants, abused drugs (amphetamine,methamphetamine, cannabinoids, cocaine and opiates) and antibodies toany number of viruses or bacteria outlined below.

As will be appreciated by those in the art, a large number of analytesmay be detected using the present methods; basically, any target analytefor which a binding ligand, described below, may be made may be detectedusing the methods of the invention.

In one embodiment, the target analytes are nucleic acids. By “nucleicacid” or “oligonucleotide” or grammatical equivalents herein means atleast two nucleotides covalently linked together. A nucleic acid of thepresent invention will generally contain phosphodiester bonds, althoughin some cases, as outlined below, nucleic acid analogs are included thatmay have alternate backbones, comprising, for example, phosphoramide(Beaucage et al., Tetrahedron 49(10):1925 (1993) and references therein;Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl et al., Eur. J.Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res. 14:3487(1986); Sawai et al, Chem. Lett. 805 (1984), Letsinger et al., J. Am.Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta 26:14191986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437(1991); and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al.,J. Am. Chem. Soc. 111:2321 (1989), O-methylphophoroamidite linkages (seeEckstein, Oligonucleotides and Analogues: A Practical Approach, OxfordUniversity Press), and peptide nucleic acid backbones and linkages (seeEgholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed.Engl. 31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson et al.,Nature 380:207 (1996), all of which are incorporated by reference).Other analog nucleic acids include those with bicyclic structuresincluding locked nucleic acids, Koshkin et al., J. Am. Chem. Soc.120:13252-3 (1998); positive backbones (Denpcy et al., Proc. Natl. Acad.Sci. USA 92:6097 (1995); non-ionic backbones (U.S. Pat. Nos. 5,386,023,5,637,684, 5,602,240, 5,216,141 and 4,469,863; Kiedrowshi et al., Angew.Chem. Intl. Ed. English 30:423 (1991); Letsinger et al., J. Am. Chem.Soc. 110:4470 (1988); Letsinger et al., Nucleoside & Nucleotide 13:1597(1994); Chapters 2 and 3, ASC Symposium Series 580, “CarbohydrateModifications in Antisense Research”, Ed. Y. S. Sanghui and P. Dan Cook;Mesmaeker et al., Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffset al., J. Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743(1996)) and non-ribose backbones, including those described in U.S. Pat.Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series580, “Carbohydrate Modifications in Antisense Research”, Ed. Y. S.Sanghui and P. Dan Cook. Nucleic acids containing one or morecarbocyclic sugars are also included within the definition of nucleicacids (see Jenkins et al., Chem. Soc. Rev. (1995) pp 69-176). Severalnucleic acid analogs are described in Rawls, C & E News Jun. 2, 1997page 35. All of these references are hereby expressly incorporated byreference. These modifications of the ribose-phosphate backbone may bedone to facilitate the addition of ETMs, or to increase the stabilityand half-life of such molecules in physiological environments.

As will be appreciated by those in the art, all of these nucleic acidanalogs may find use in the present invention. In addition, mixtures ofnaturally occurring nucleic acids and analogs can be made; for example,at the site of conductive oligomer or ETM attachment, an analogstructure may be used. Alternatively, mixtures of different nucleic acidanalogs, and mixtures of naturally occuring nucleic acids and analogsmay be made.

The nucleic acids may be single stranded or double stranded, asspecified, or contain portions, of both double stranded or singlestranded sequence. The nucleic acid may be DNA, both genomic and cDNA,RNA or a hybrid, where the nucleic acid contains any combination ofdeoxyribo- and ribo-nucleotides, and any combination of bases, includinguracil, adenine, thymine, cytosine, guanine, inosine, xathaninehypoxathanine, isocytosine, isoguanine, etc. One embodiment utilizesisocytosine and isoguanine in nucleic acids designed to be complementaryto other probes, rather than target sequences, as this reducesnon-specific hybridization, as is generally described in U.S. Pat. No.5,681,702. As used herein, the term “nucleoside” includes nucleotides aswell as nucleoside and nucleotide analogs, and modified nucleosides suchas amino modified nucleosides. In addition, “nucleoside” includesnon-naturally occurring analog structures. Thus for example theindividual units of a peptide nucleic acid, each containing a base, arereferred to herein as a nucleoside.

Thus, in one embodiment, the target analyte is a target sequence. Theterm “target sequence” or “target nucleic acid” or grammaticalequivalents herein means a nucleic acid sequence on a single strand ofnucleic acid. The target sequence may be a portion of a gene, aregulatory sequence, genomic DNA, cDNA, RNA including mRNA and rRNA, orothers. As is outlined herein, the target sequence may be a targetsequence from a sample, or a secondary target such as a product of anamplification reaction, etc. It may be any length, with theunderstanding that longer sequences are more specific. As will beappreciated by those in the art, the complementary target sequence maytake many forms. For example, it may be contained within a largernucleic acid sequence, i.e. all or part of a gene or mRNA, a restrictionfragment of a plasmid or genomic DNA, among others. As is outlined morefully below, probes are made to hybridize to target sequences todetermine the presence or absence of the target sequence in a sample.Generally speaking, this term will be understood by those skilled in theart. The target sequence may also be comprised of different targetdomains; for example, a first target domain of the sample targetsequence may hybridize to a capture probe or a portion of captureextender probe, a second target domain may hybridize to a portion of anamplifier probe, a label probe, or a different capture or captureextender probe, etc. The target domains may be adjacent or separated asindicated. Unless specified, the terms “first” and “second” are notmeant to confer an orientation of the sequences with respect to the5′-3′ orientation of the target sequence. For example, assuming a 5′-3′orientation of the complementary target sequence, the first targetdomain may be located either 5′ to the second domain, or 3′ to thesecond domain.

Suitable target analytes include biomolecules associated with: (1)viruses, including but not limited to, orthomyxoviruses, (e.g. influenzavirus), paramyxoviruses (e.g respiratory syncytial virus, mumps virus,measles virus), adenoviruses, rhinoviruses, coronaviruses, reoviruses,togaviruses (e.g. rubella virus), parvoviruses, poxviruses (e.g. variolavirus, vaccinia virus), enteroviruses (e.g. poliovirus, coxsackievirus),hepatitis viruses (including A, B and C), herpesviruses (e.g. Herpessimplex virus, varicella-zoster virus, cytomegalovirus, Epstein-Barrvirus), rotaviruses, Norwalk viruses, hantavirus, arenavirus,rhabdovirus (e.g. rabies virus), retroviruses (including HIV, HTLV-1 and-11), papovaviruses (e.g. papillomavirus), polyomaviruses, andpicomaviruses, and the like; and (2) bacteria, including but not limitedto, a wide variety of pathogenic and non-pathogenic prokaryotes ofinterest including Bacillus; Vibrio, e.g. V. cholerae; Escherichia, e.g.Enterotoxigenic E. coli, Shigella, e.g. S. dysenteriae; Salmonella, e.g.S. typhi; Mycobacterium e.g. M. tuberculosis, M. leprae; Clostridium,e.g. C. botuliniin, C. tetani, C. difficile, C. perfringens;Cornyebacterium, e.g. C. diphtheriae; Streptococcus, S. pyogenes, S.pneumoniae; Staphylococcus, e.g. S. aureus; Haemophilus, e.g. H.influenzae; Neisseria, e.g. N. meningitidis, N. gonorrhoeae; Yersinia,e.g. G. lamblia Y. pestis, Pseudomonas, e.g. P. aeruginosa, P. putida;Chlamydia, e.g. C. trachonmatis; Bordetella, e.g. B. pertussis;Treponema, e.g. T. palladium; and the like.

Other suitable target analytes include, but are not limited to, (1)enzymes (and other proteins), including but not limited to, enzymes usedas indicators of or treatment for heart disease, including creatinekinase, lactate dehydrogenase, aspartate amino transferase, troponin T,myoglobin, fibrinogen, cholesterol, triglycerides, thrombin, tissueplasminogen activator (tPA); pancreatic disease indicators includingamylase, lipase, chymotrypsin and trypsin; liver function enzymes andproteins including cholinesterase, bilirubin, and alkaline phosphotase;aldolase, prostatic acid phosphatase, terminal deoxynucleotidyltransferase, and bacterial and viral enzymes such as HIV protease; (2)hormones and cytokines (many of which serve as ligands for cellularreceptors) such as erythropoietin (EPO), thrombopoietin (TPO), theinterleukins (including IL-1 through IL-17), insulin, insulin-likegrowth factors (including IGF-1 and -2), epidermal growth factor (EGF),transforming growth factors (including TGF-n and TGF-P), human growthhormone, transferrin, epidermal growth factor (EGF), low densitylipoprotein, high density lipoprotein, leptin, VEGF, PDGF, ciliaryneurotrophic factor, prolactin, adrenocorticotropic hormone (ACTH),calcitonin, human chorionic gonadotropin, cotrisol, estradiol, folliclestimulating hormone (FSH), thyroid-stimulating hormone (TSH), leutinzinghormone (LH), progeterone and testosterone; and (3) other proteins(including (Y-fetoprotein, carcinoembryonic antigen CEA, cancer markers,etc.).

Suitable target analytes include carbohydrates, including but notlimited to, markers for breast cancer (CA15-3, CA 549, CA 27.29),mucin-like carcinoma associated antigen (MCA), ovarian cancer (CA125),pancreatic cancer (DE-PAN-2), prostate cancer (PSA), CEA, and colorectaland pancreatic cancer (CA 19, CA 50, CA242).

Other suitable target analytes include metal ions, particularly heavyand/or toxic metals, including but not limited to, aluminum, arsenic,cadmium, selenium, cobalt, copper, chromium, lead, silver and nickel.

In one embodiment, the methods of the invention are used to detectpathogens such as bacteria. In this embodiment, target sequences includerRNA, as is generally described in U.S. Pat. Nos. 4,851,330; 5,288,611;5,723,597; 6,641,632; 5,738,987; 5,830,654; 5,763,163; 5,738,989;5,738,988; 5,723,597; 5,714,324; 5,582,975; 5,747,252; 5,567,587;5,558,990; 5,622,827; 5,514,551; 5,501,951; 5,656,427; 5.352.579;5,683,870; 5,374,718; 5,292,874; 5,780,219; 5,030,557; and 5,541,308,all of which are expressly incorporated by reference.

In one embodiment nucleic acid sequencing methods are used. Sequencingmethods are described in U.S. Ser. Nos. 09/626,096, filed Jul. 26, 2000,09/847,113, filed May 1, 2001, 10/137,710, filed Apr. 30, 2002,10/336,255, filed Jan. 2, 2003 and 10/823,502, filed Apr. 12, 2004, allof which are expressly incorporated herein by reference.

As will be appreciated by those in the art, a large number of analytesmay be detected using the present methods; basically, any target analytefor which a binding ligand, described below, may be made may be detectedusing the methods of the invention. While many of the techniquesdescribed below exemplify nucleic acids as the target analyte, those ofskill in the art will recognize that other target analytes can bedetected using the same systems.

If required, the target analyte is prepared using known techniques. Forexample, the sample may be treated to lyse the cells, using known lysisbuffers, electroporation, etc., with purification and/or amplificationas needed, as will be appreciated by those in the art. When the targetanalyte is a nucleic acid, the target sequence may be amplified asrequired; suitable amplification techniques are outlined in PCTUS99/01705, hereby expressly incorporated by reference. In addition,techniques to increase the amount or rate of hybridization can also beused; see for example WO 99/67425 and U.S. Ser. Nos. 09/440,371 and60/171,981, all of which are hereby incorporated by reference.

The samples comprising the target analytes can be added to the fluidicsdevices described herein. By “fluidics device” is meant devicecomprising a substrate, at least one channel, inlet ports and outletports as well as valves. The fluidics device of this disclosure can takeon numerous configurations.

By “cartridge” herein is meant a casing or housing for the biochip. Asoutlined herein, and as will be appreciated by those in the art, thecartridge can take on a number of configurations and can be made of avariety of materials. Suitable materials include, but are not limitedto, fiberglass, teflon, ceramics, glass, silicon, mica, plastic(including acrylics, polystyrene and copolymers of styrene and othermaterials, polypropylene, polyethylene, polybutylene, polycarbonate,polyurethanes, Teflon™, and derivatives thereof, etc.), etc.Particularly preferred cartridge materials are plastic (includingpolycarbonate and polyproplylene) and glass.

As will be appreciated by those in the art, the cartridge can comprise anumber of components, including reaction chambers, inlet and outletports, heating elements including thermoelectric components, RFantennae, electromagnetic components, memory chips, sealing componentssuch as gaskets, electronic components including interconnects,multiplexers, processors, etc.

In one embodiment, the cartridge comprises a reaction chamber.Generally, the reaction chamber comprises a space or volume that allowsthe contacting of the sample to the biochip array. The volume of thereaction chamber can vary depending on the size of the array and theassay being done. In general, reaction chamber ranges from 1 nL to about1 mL, with from about 1 to about 250 .mu.l being preferred and fromabout 10 to about 100 .mu.l being especially preferred. In someembodiments, to avoid the introduction of air bubbles into the reactionchamber (which can be disruptive to detection), the reaction chamber isless than the size of the sample to be introduced, to allow a slightoverflow and thus ensure that the reaction chamber contains little or noair.

In one embodiment, the biochip cartridge can be configured to includeadditional chambers that can used for any number of different reactions,such as sample preparation, cell lysis, rare targetcapture/concentration, sample clean-up, nucleic acid amplification,including PCR, post-amplification clean-up, sample concentration,reagent storage, mixing baffles/devices, etc. In other embodiments, thereaction chamber may be configured for other types of reactions asgenerally described below.

In one embodiment, the biochip cartridge reaction chamber is configuredto include at least one nucleic acid amplification chamber. However,multiple amplification chambers may be used. That is, a cartridge maycomprise from about 1 to about 10 or more chambers, with 2, 3, 4, 5, 6,7, 8 or 9 also being preferred.

In one embodiment, the biochip cartridge reaction chamber is configuredto include at least one PCR chamber. However, multiple PCR chambers maybe used. That is, a cartridge may comprise from about 1 to about 10 ormore chambers, with 2, 3, 4, 5, 6, 7, 8 or 9 also being preferred.

In one embodiment, the chamber of the cartridge should be made frombiocompatible materials. In particular, materials that provide a surfacethat retards the non-specific binding of biomolecules, e.g. a “nonsticky” surface, are preferred. For example, when the reaction chamberis used for PCR or amplification reactions a “non sticky” surfaceprevents enzymatic components of the reaction mixture from sticking tothe surface and being unavailable in the reaction. In addition, thebiocompatible properties of the chamber may be improved by minimizingthe surface area.

Biocompatible materials include, but are not limited to, plastic(including acrylics, polystyrene and copolymers of styrene and othermaterials, polypropylene, polyethylene, polybutylene, polyimide,polycarbonate, polyurethanes, Teflon.™., and derivatives thereof, etc.)Other configurations include combinations of plastic and printed circuitboard (PCB; defined below). For example at least one side of the chamberis printed circuit board, while one or more sides of the chamber aremade from plastic. In one embodiment, three sides of the chamber aremade from plastic and one side is made from printed circuit board. Inaddition, the chambers, channels, valves, pumps, etc. of the systemsdescribed herein may be coated with a variety of materials to reducenon-specific binding. These include proteins such as caseins andalbumins (bovine serum albumin, human serum albumin, etc.), parylene,other polymers, etc.

In one embodiment, the reaction chamber of the cartridge comprises aninlet port for the introduction of the sample to be analyzed. Dependingon the reaction being run, multiple inlet ports may be used, that mayfeed from a variety of storage chambers or from the outside of thechamber. The inlet port may optionally comprise a seal to prevent orreduce the evaporation of the sample or reagents from the reactionchamber. In one embodiment, the seal comprises a gasket, or valvethrough which a pipette or syringe can be pushed. The gasket or valvecan be rubber or silicone or other suitable materials, such as materialscontaining cellulose.

The reaction chamber can be configured in a variety of ways. In oneembodiment, the reaction chamber is configured to minimize theintroduction or retention of air bubbles or other sample impurities.Thus for example, assuming that the cartridge is held in an uprightangle, the inlet port allows the flow of fluid sample into the “bottom”of the reaction chamber, to allow the escape of air or fluid through the“top” of the reaction chamber, for example through an outlet port. Thusthe fluid sample flows up into the reaction chamber and contacts thearray. Thus, in one embodiment, the reaction chamber further comprisesan outlet port to allow air or excess sample to exit the reactionchamber. In some embodiments, the outlet port vents to either a wastestorage well, as is further described below, to an external surface ofthe chip or cartridge, or, in one embodiment, back into the inlet port.Thus for example one embodiment utilizes a system wherein the exit portvents to the inlet port, preferably above the point of loading. Forexample, when a pipette is used to load the cartridge, the tip of thepipette extends below the exit port, such that air from the exit port isnot introduced into the reaction chamber. In addition, the materials ofthe cartridge housing and biochip can be chosen to be similar inhydrophobicity or hydrophilicity, to avoid the creation of air bubbles.

In one embodiment, an anti-siphon vent is used to prevent liquid frombeing sucked back into a chamber as a result of the negative pressuregenerated when an air pump heater is turned off. For example, aanti-siphon vent comprising a paraffin valve and an open port can beconstructed between the reaction chamber and an air pump.

In addition, in one embodiment, the reaction chamber/inlet and/or outletports optionally include the use of valves. For example, asemi-permeable membrane or filter may be used, that preferentiallyallows the escape of gas but retains the sample fluid in the chamber.For example, porous teflons such as Gortex™ allow air but not fluids topenetrate.

In one embodiment, a reaction chamber in the biochip cartridge (such asa PCR chamber) has one or more valves controlling the flow of fluidsinto and out of the chamber. The number of valves in the cartridgedepends on the number of channels and chambers. Alternatively, thebiochip cartridge is designed to include one or more loading ports orvalves that can be closed off or sealed after the sample is loaded. Itis also possible to have multiple loading ports into a single chamber;for example, a first port is used to load sample and a second port isused to add reagents. In these embodiments, the biochip cartridge mayhave a vent. The vent can be configured in a variety of ways. In someembodiments, the vent can be a separate port, optionally with a valve,that leads out of the reaction chamber. Alternatively, the vent may be aloop structure that vents liquid and/or air back into the inlet port.

As will be appreciated by those in the art, a variety of differentvalves may be used. Microvalves can be categorized into two major types:passive microvalves (without actuation) and active microvalves (with anactuation). Generally, active microvalves couple a flexible diaphragm toa thermopneumatic, piezoelectric, electrostatic, electromagnetic,bimetallic actuator. Additional valves find use in the invention and aredescribed in more detail in US Pub. No. 2007/0098600 which is expressllyincorporated herein by reference for disclosure describing valves.

Accordingly, in a first aspect, the invention features a fluidicsdevice, preferably multilaminate, having a diaphragm and a plurality ofcheck valves contiguous with said diaphragm through one or more flowchannels. The diaphragm is acted upon by a pump, which in one embodimentis extraneous to the device itself but nevertheless engageablyinterfaceable therewith. In one embodiment the check valves are passive“bridge” valves as described herein, which essentially comprise more orless parallel slits in a flexible laminate sheet which can allowdisplacement of the section therebetween upon application of force,thereby routing fluid flow along a new path, in one embodiment, in adirection more or less parallel to the laminate portion flanking theslits. In one embodiment the system is pump or vacuum-driven.

By “multilaminate” is meant prepared from multiple, e.g. more than one,layers. Polyethyleneterepthalate (PET) and etchedpolytetrafluoroethylene (PTFE) find particular use as constructionmaterials, but other materials as discussed herein can also be used,with the overall multilaminate comprising a single material or multipledifferent materials. In general, as described herein, at least 2, 3, 4,5 or more layers are used, as is shown in the figures. Generally, one ormore of the layers have vias, e.g. shaped holes therein, such that whensandwiched between two additional layers (e.g. a “top” and a “bottom”)they form the channels of the chips as described herein. Similarly, oneof the layers generally includes the array of electrodes (which asoutlined herein can be within a detection chamber as a rectilineararray, within a detection chamber comprising a serpentine (or othergeometries) channel, etc.), which is laminated to a layer defining achannel. As described herein, the layers may be attached in a widevariety of ways, including adhesives (pressure sensitive, heatsensitive, etc.). In addition, as outlined herein, the individual layerscan also contain features for attachment, like posts or pegs that coupleto corresponding holes in another layer.

By “diaphragm” is meant a flexible seal that flexes in a positive andnegative fashion. In one emboidment the diaphragm is positioned in afirst chamber or separates multiple chambers. In one embodiment thediaphragm is within an enclosed system. Thus, upon movement of thediaphragm ni a positive or negative direction, either air or materialswithin the enclosed system move in response to the movement of thediaphragm. In one embodiment the diaphragm includes a magnet. Thisallows for movement of the diaphragm by controlling movement of themagnet, which can be accomplished electrically.

By “flow channels” is meant a channel through which a liquid flows inthe cartridge of this disclosure. The dimensions of the flow channel aresignificant in that the linear speed of the fluid over the electrodes isdependent on the channel cross section. Additionally, the channel heightand width must be great enough to allow any bubbles to freely flowthrough the channel and eventually be trapped/cleared in the reservoir.Therefore, it is important to design a channel that is small enough toprovide sufficient linear velocity but not too restrictive with respectto bubble movement. Another consideration is that the lower channeldimensions will utilize less analyte solution, often a desiredcharacteristic. Channel widths of may be from 0.020-0.100″, morepreferably, 0.025-0.080″ and more preferably from 0.030-0.060″. Channelheights may be from 0.002-0.020″, more preferably from 0.004-0.015″ andmore preferbly from 0.006-0.015″. For example, channel widths0.030-0.060″ and heights of 0.006″-0.014″ were tested as compatibleranges, with the preferred dimensions being a width of 0.040″ and heightof 0.010″.

Valves are described above. In one embodiment the check valve is a“bridge valve” as decribed herein. In some embodiments, the bridgevalves are contained in the same laminar piece or layered composite asthe diaphragm.

In one embodment the diaphragm is in a first chamber such as a reactionchamber and a secone chamber such as a detection chamber may beseparate. In some embodiments channels connect the first and secondchambers as described herein. Alternatively, the channel itself is usedfor detection.1 In this embodiment the detection chamber or the channel,when it is used for detection, includes ligands for binding targetanalytes as described herein. Also as described herein the ligands mayform an array on a substrate in the detecion chamber or channel.

The channels are preferably defined by two or more laminate layers andpreferably elongated, most preferably serpentine. Preferably, inside thechannel are surfaces containing affixed ligand(s), which in turnabstract or bind specific complement, anti-ligand or analyte from afluid sample that is pumped through the channel and across thosesurfaces. Preferably the abstracting surfaces are configured in adimension that does not waste surface area space, the effect of which isto conserve volume and sensitivity per sample volume, and thereby allowfor or facilitate miniaturization.

There are two primary means by which fluid can be moved in the biochipcartridge. These are: (1) through the use of a pump that pushes thefluid in or out; or, (2) by suction that pulls fluid in or out of thechamber.

Generally, a device such as a moving piston is used to create suction,however cooling of gases, vacuum chambers and gas consuming reactionscan be used. When suction is used to move liquid in or out of thechamber, a vacuum may be created elsewhere in the system.

In some embodiment the pump can be on the chip or off the chip. By “onchip” is meant that the pump is integral to the cartridge itself. by“off chip” is meant that the chip is separate from and not integral tothe chip. Basically, two major groups of pumps, classified based ondifferent pump mechanisms (i.e., actuation), can be use in the presentinvention: membrane actuated (i.e., mechanical) and non-membraneactuated pumps. Membrane actuated pumps can be further divided intothree types: piezoelectric, electrostatic, and thermopneumatic.Non-membrane pumping principles include electrohydrodynamic,electroosmotic, traveling wave, diffuser, bubble, surface wetting,rotary, etc.

In one embodiment, an “air pump” is used to move the liquid out of thePCR chamber. In this embodiment, a chamber of air is incorporated in thechip with an “on chip” heater. When the heater is turned on, the air inthe chamber expands according to PV=nRT. In some embodiments, the airpump is incorporated into the cartridge.

Preferably, heaters (as are also described below) are incorporated intothe middle of the chip. In some embodiments, more than one heater isincorporated in a chip to create “heater zones”. Air chambers or pocketsare located over the heater zones. The air chambers are connected to thereaction chamber via a channel that runs up to the top of the reactionchamber with a valve or a plug blocking it off. When the air is heated,it expands. The resulting build up in pressure forces the valve or plugto move out of the way, thereby forcing the liquid out of the chambervia an outlet port.

Other ways of moving liquid out of the reaction chamber or reactionchamber include using a low boiling liquid in place of air. In thisembodiment, the low boiling liquid expands when heated and displaces theliquid contained in the reaction chamber. Alternatively, a chemicalreaction may be used to move liquid out of the reaction chamber. Forexample, the chemical reaction used to expand car air bags may be usedto move liquid out of the reaction chamber, or other reactions in whichgases are generated.

Other types of pumps that can be used include syringe driven pumps.These pumps can be actuated either by expanding air behind the syringeor by mechanical means. For example, TiNi alloys, nitinol wire, or“shape memory metals” can be used to mechanically actuate a syringedriven pump. By “TiNi alloys”, “nitinol wire” or “shape memory metals”herein is meant materials that when heated above a certain transitiontemperature contract (i.e., usually up to 3 to 5% over the originallength of the metal), thereby changing shape. Other materials thatchange shape upon heating include shape memory plastics.

Pumps also may be created using spring loaded pistons. In thisembodiment, a spring that can be released is compressed or restrainedwithin the body of the cartridge. For example, wax may be used to hold aspring in its compressed state. Upon heating, the wax is melted, and thespring is released, thereby generating sufficient force to move a pistonand displace liquid. Other versions include incorporating materials thatchange from solids to liquids at a given transition temperature, ormoving a mechanical blockade from the spring's pathway.

Pumps that utilize PZT driven actuations are also known and may beincorporated int this invention. By “PZT” herein is meant a materialcomprised of lead, zirconium and titanium which upon application of avoltage undergoes a rearrangement of the crystal lattice and generates aforce and a displacement. This so called piezoelectric effect can beused to constrict and expand a pump chamber and result in a net movementof liquid. Other materials like shape memory alloys that under a changein shape upon application of a current such that the temperature of themetal is raised above a certain transition temperature can also be used.

In addition, commercially available micro pumps may be used in to moveliquid from one location to another in the cartridge. Examples ofcommercially available pumps include, moulded plastic micro pumpsavailable from IMM (see liganews@imm.uni-mainz.de), thin film shapealloy microactuators (TiNi Alloy Company, San Leandro, Calif.), siliconmicro pumps (see M. Richter & J. Kruckow,aktorik/paper/2000jahresbericht/Paper2, 16.11.00).

In addition, based on the geometry of the chamber, air can be used topush liquid out of the reaction chamber or mix liquids within thereaction chamber. Whether the air pumps the fluid or bubbles through togenerate a mixing effect is determine by the relative size of thebubble, the geometry of the chamber/channel and the surface tension ofthe liquid. Larger air-liquid interfaces tend to favor mixing overpumping. Mixing of liquid within the biochip cartridge can occur bypumping the liquid back and forth in the biochip cartridge.

In one embodiment, flow-induced mixing is used to induce convectionalflow. Preferably, this is used in a vertical system, such that fluidgravity may be used to induce convectional flow. The convectional flowresults in bulk fluid mixing between two liquid solutions. In addition,meniscus recirculation mixing can be used to induce circulation flow(Anderson, et al., (1998) Solid-State Sensor and Actuator Workship,Hilton Head Island, June 9-11, pp 7-10; incorporated herein by referencein its entirety).

In one embodiment, mixing is accomplished by creating a thermal gradientacross a chip. For example, a thermal gradient may be created by heatingthe bottom of the chip to 65° C. and cooling the top of the cartridgecover to 10° C. This can be accomplished by placing the chip between twopeltier heaters, or by using an imbedded heater and a single peltier orother thermoelectric cooling devices.

In one embodiment, mixing is accomplished by recirculating liquid in agiven chamber using an on chip or “off chip” pump attached to a chip.

In alternative embodiments, mixing is accomplished by recirculatingliquid using a micro disk-pump, such as a plastic disk embedded with amagnetic steel bar. Rotation of the disk pump may be achieved using anexternal magnetic filed provided by a standard stirrer or custom builtwith multiple fields. See also U.S. Ser. No. 60/308,169, filed Jul. 26,2001 and a provisional application by Gallagher, et al., entitled“System and methods for mixing within a microfluidic chamber”, filedJul. 11, 2002; both of which are incorporated by reference in theirentirety.

In other embodiments, biochannel based mixing can be used to enhancehybridization rates. In this embodiment, a bubble is intentionallyintroduced into one corner of the chip. By alternately expanding andcontracting the bubble volume via the application of heat from either anin chip or off chip heat source, mixing occurs as a result of thepressure flow created by changing the volume of the bubble within thechip. Alternatively, resonance induced mixing of bubbles can be doneusing PZT devices as well.

In some embodiments, mixing may be accomplished using non-contact mixingtechnologies like that describe by Covaris, Inc.

In one embodiment, heaters are incorporated onto or into the chip, toallow “on chip” heating (in addition, as described below, “off chip”thermocontrollers within the device may also be used). In thisembodiment, the reaction chamber is designed to maximize thermalconductivity between the chamber and the heater or themocontroller.Generally, designs that minimize thermal mass (i.e., making the surfaceof the chamber in contact with the heat source as thin as possible),impose certain geometric constraints to ensure the complete removal ofliquid from the chamber, incorporate materials that are good thermalconductors (i.e., metals), and thermally isolate the chamber from therest of the chip are preferred. Often one makes a trade off betweenminimizing surface to volume ratios to reduce surface area for thenon-specific binding of biological components and maximizingsurface-to-volume ratis in order to obtain rapid heat transfer rates forheating and cooling.

In one embodiment, air pockets or vents are used to thermally isolatethe amplificationchamber from the rest of the chip. That is, the thereis a break in the continuity of the cartridge around the amplificationchamber.

In one embodiment, thermally conductive materials are incorporated intoor below the reaction chamber, forming hybrid chambers. For example, byusing “layers” of different materials, effective heaters areconstructed. Thus for example, one embodiment utilizes one or moreresistive heaters in the form of resistive metallic inks can be appliedto a first layer of PC board. These heaters are powered byinterconnects. In one embodiment, a thin sheet of a thermally conductivematerial, preferably a metal such as copper, is applied, to allow evenheat distribution. In one embodiment, the copper layer is then coatedwith a thin layer of biocompatible material, such as plastic. See FIG.10A.

The total thickness of the hybrid chamber may vary from a few microns tomillimeter dimension. A preferred thickness is approximately 200microns.

In one embodiment, multiple thermal heaters are incorporated into thedevice to allow for the creation of multiple thermal zones. Thetemperature in the respective zones is maintained via either active orpassive control. Frequently, thethermal connectivity of the cartridgematerials are taken into account during the design. In one embodiment, achip may contain a thermal heater in the detection chamber of thecartridge in order to maintain the temperature of the detection chamberas well as constructing unique temperature zones in another part ofdevice. In one embodiments, these temperature zones may be maintained toallow an enzymatic reaction to run efficiently. In another embodiment,multiple temperature zones may be maintained to simulate thetemperatures normally used during PCR heat cycling. In order to effectthe necessary temperature, the liquid can be maintained stationary andthe temperature of the amplification chamber cycled (i.e. 95-55-72),alternatively, the liquid can be pumped over different temperature zonesin order to obtain heat cycling. This embodiment can be realized indifferent material substrates such as glass, plastic, ceramic and PCB.

Similarly, there may be portions of the substrate that require heating,and those that do not. Thus more than one heater may be incorporatedinto the substrate. Similarly, these thermal zones may or may not bethermally isolated from other parts of the substrate. For example, PCboard is significantly thermally insulative, and thus just puttingdistance between the heaters and thermal zones and the areas of thesubstrate that do not require heating may be sufficient. In otherembodiments, thermally insulative materials may be incorporated. Forexample, when the substrate is a ceramic material, thermal isolation maybe accomplished by cutting out sections of the ceramic substrate suchthat solid regions of ceramic are separated from one another by a “cutout”.

Other embodiments include the incorporation of temperature sensors intothe substrate such that the temperature throughout the board can bemonitored. In one embodiment, temperature sensors are created usingresistive devices, including silicon diodes. Other embodiments includethe use of capillary thermostats and limiters, such as those offered byThermodisc.

As will be appreciated by those in the art, there are a variety ofreaction chamber geometries which can be used in this way. Generallyhaving the intersection of the inlet port and the reaction chamber be atthe “bottom” of the cartridge, with a small aperture, with the reactionchamber widening, is preferred. In addition, the “top” of the reactionchamber may narrow, as well. Thus, preferred embodiments for the sizeand shape of the reaction chamber allow for smooth loading of thereaction chamber. Preferred embodiments utilize reaction chambergeometries that avoid the use of sharp corners or other components thatserve as points for bubble formation.

In addition, in some embodiments, the reaction chamber can be configuredto allow mixing of the sample. For example, when a sample and a reagentare introduced simultaneously or separately into the chamber, the inletport and/or the reaction chamber can comprise weirs, channels or othercomponents to maximize the mixing of the sample and reagent. Inaddition, as is outlined below, the reaction may utilize magnetic beadsfor mixing and/or separation.

In one embodiment, the cartridge comprises a sealing and/or ventingmechanism to prevent the cartridge from exploding due to a build up inpressure during a reaction, or to prevent leakage of the sample orreagents onto other parts of the substrate, particularly (in the case ofelectronic detection) onto electronic interconnects. As will beappreciated by those in the art, this may take on a variety of differentforms. In one embodiment, there is a gasket between the biochipsubstrate comprising the array and the cartridge, comprising sheets,tubes or strips. Alternatively, there may be a rubber or silicone stripor tube used; for example, the housing may comprise an indentation orchannel into which the gasket fits, and then the housing, gasket andchip are clamped together. Furthermore, adhesives can be used to attachthe gasket to the cartridge, for example, a double sided adhesive can beused; for example, silicone, acrylic and combination adhesives can beused to attach the gasket to the biochip, which is then clamped into thecartridge as described herein.

In embodiments where the surfaces are electrodes, each electrode surfacepreferably occupies a majority of the channel width and is positioned inseries relative to other electrodes, with a channel height thereoverthat allows fluid to flow thereacross through the channel.

In some embodiments, the individual laminate pieces are substantiallyplanar and “stack” to thereby form the multilaminate device and internalfeatures in operable form. In other embodiments, the individual laminatepieces are substantially nonplanar or modular, and still cooperativelystack or interface, e.g., like Ruffles® or Pringles® potato chips.

In a second aspect, the invention features a method of constructing amultilaminate fluidics device by mating/conjoining the individuallaminate pieces noted above. This is preferably accomplished by stackingand fusing or otherwise sealing the individual pieces together tothereby form a device having one or more functional chambers, channels,diaphrams and/or valves, etc. therein.

In yet a third aspect, the invention features a method of using amultilaminate fluidics device as above by adding a fluid sample theretoand interfacing with a pump mechanism (e.g., syringe pneumatic,hydrolic, thermal, or electromechanical) and magnetic, electronic, orother detection device (e.g., colorimetric, electrochemical, isotopic,densitometric, etc.), and prectifying flow through the device so thatthe sample can be acted upon or analyzed, e.g., by a laser or detectorin one or more downstream channels or chambers.

In some embodiments, flow is preferably of a two-stroke design whereinthe diaphragm is periodically moved back and forth or up and down toactuate/rectify directional fluid flow across cooperating tandem valveshaving the diaphragm fluidly coupled therebetween. In operation, onevalve is substantially closed when the other is substantially open,depending on stroke, and vice-versa.

In some embodiments the device is a disposable one, and reversiblyengageable with a pump and/or electronic stimulation or detectioncomponent that can be used over and over again, e.g., a laser and/or anoptical, voltametric, amperometric, and/or thermal reader.

In some embodiments, the device is used for in vitro diagnostics.

In some embodiments, the device is used with or has integrated afiltration, purification, separation, and/or mixing means as known inthe art.

In a fourth aspect the invention features a single laminar sheet bearingone or more of a diaphragm, valving, and/or channels. This sheet can bea component of the first aspect. In embodiments, the sheet is formed ofeither PET, polypropylene, ultra high molecular weight polyethylene, lowdensity polyethylene, high density polyethylene, linear low densitypolyethylene and/or Teflon®, and is preferably 5 mm or less inthickness, more preferably 2 mm or less in thickness, and mostpreferably 1 mm or less in thickness. These sheets may be present aspart of a large roll of individual sheets of identical or complementarydimension. The sheets may be individually machined, molded, stamped,chemically-etched and/or laser ablated to carry the individual featuresnoted. The individual sheets may also be perforated or otherwiserendered separable from the roll to be thereafter incorporated into alarger multilaminate fluidics device according to the invention. One canreadily envision an automated or semi-automated procedure whereby auniform roll of laminate undergoes stamping, machining, molding,etching, welding and/or ablation to endow microfluidics features in avolume batching format using standard methodologies and capabilitiesknown in the art.

In a fifth aspect the invention features a device having inlet andoutlet ports that are joined to effectively re-circulate sample whilesimultaneously facilitating gas removal and thereby facilitatingmolecular diffusion and efficiency of the microfluidic system. Thisaspect can be combined with any of the preceding aspects and embodimentsas appropriate, e.g., by suitable injection molding, machining,stamping, etching, ablation, and mated sealing of individual laminatesusing adhesives, gaskets, clamps, solvent bonding, ultrasonic welding,etc. Preferably, gas removal is accomplished by circulating the fluidsample past a bubble trap or enclosed gaseous environment located abovethe fluid flow path.

In some embodiments, the multilaminate device processes (or are designedto process) no more than 1 mL of fluid/analyte sample at a time. In someother embodiments, no more than 150 uL of fluid is processed at a time.In still others, preferably no more than about 100 uL or less isprocessed at a time.

In multilaminate embodiments, the individual pieces may optionally beheld together and sealed by pressure sealing adhesive sheets, welding,and/or using conformable gasket-like material such as silicone orsilicone sheeting.

In some embodiments, one or more channels or chambers are formed bygrooves, cuts or recesses in one or more of the cooperating laminatesheets, e.g., as provided by a die-stamp, chemical etching or laserablation technique, as those techniques are commonly understood in theart.

In some embodiments, biological binding partners such as proteins,peptides, antibodies, nucleic acid and polynucleotides/oligonucleotidesare preferably attached to surfaces in the system, e.g., on electrodes,which are preferably contained in some of the channels or chambers thatreceive the liquid samples upon flow through the device. Preferablythose samples are recirculated, which improves binding efficiency andresult by simultaneously augmenting diffusion and effecting mixing,washing and shear strain to overcome problems associated with laminarflow.

A continuous flow rate of about 10-40 uL/sec in connection with thedimensions used herein has been found to be optimal, but pulsed flow isalso envisoned to work. These principles need not be tied tomultilaminate devices alone, but can be adopted for any device and withlike effect.

Preferred attachment means for the binding partners are by adsorption orself assembling monolayer derivatization and addition/spotting to/of thesurfaces on which they are affixed, e.g., electrode(s).

In some embodiments, the devices are preferably made of a transparent ortranslucent material that permits visualization of the circulation andrecirculation of fluid sample.

In some embodiments, syringes or pneumatic pumps or other means maydrive the system, which may or may not be electromechanical in nature.

In some embodiments, the individual cartridges are “stacked” andoriented such that a set pitch exists between individual inlet sampleports in neighboring cartridges. This allows for convenient use ofmulti-well pipetting devices and the like to load samples. A commonpitch in the industry for this is ˜9 mm.

In some embodiments, channels are wider than tall. In some embodiments,channel dimensions range from about 0.030″ to about 0.060″ in width and0.006″ to about 0.014″ in height.

In some alternating laminate and adhesive layer embodiments, individuallayer thicknesses range of from about 0.0005″ to about 0.010″. In somepreferred embodiments, laminate layers are thicker than adhesive layers.In some embodiments, laminate layers range from about 0.0005″ to about0.030″ thickness and adhesive layers from about 0.001″ to about 0.003″thickness.

By “substantially parallel” is meant not perpendicular to one another.

Other aspects and embodiments will be apparent to one of ordinary skillin the art from the background documents, drawings, detaileddescription, and claims to follow.

The individual aspects and embodiments of the invention can be combinedas appropriate in any combination. Advantages from the combinationsinclude, as appropriate for a given aspect/embodiment: lower cost, easeof fabrication and mass fabrication, ease of reproducibility, improvedflow, improved mixing, and elimination or minimization of gaseous bubbleformation that would otherwise obstruct sample flow, and facilitatedanalyte diffusion and electrolyte conductivity.

Ease of Use

As the diagnostic testing environment becomes more decentralized (fromlarge central labs to smaller hospital labs, to patient bedsides, andinto the field), highly-trained and specialized operators become morescarce. Ease of use is a key criterion for successful implementation insuch an environment. This invention includes several features thataddress this need.

One way to make a system easier to use is to incorporate more“intelligence”, which is most effective when software is provided withuseful information. Incorporating inexpensive information storage intothe cartridge in a format that can be read from and written to is quiteadvantageous. Barcodes are an example of media that provide read-onlystorage that may transfer information from the cartridge manufacturer tothe instrument, e.g. identification of what test protocol should be runor what the cartridge expiration date is. Read-only systems used inconjunction with instrument databases further allow the association offixed identification information (e.g. a cartridge serial number) withdata unknowable at manufacture (such as the specific locale andinstrument used for testing, the testing status, and the reportedresults). Media such as EEPROMs and some RFID formats have the addedability to write to the cartridge, which allows independence from adatabase and permits any instrument to retrieve the information withoutrequiring networked access to a central server. (An “EEPROM” is shortfor Electronically Erasable Programmable Read-Only Memory, which is anon-volatile storage chip used in computers and other devices to storesmall amounts of volatile (configuration) data. EEPROMs come in a rangeof capacities from a few bytes to over 128 kilobytes and are typicallyused to store configuration parameters, and in modern computers theyreplace the hitherto common CMOS nonvolatile BIOS memory).

Another way to drive ease of use is to make a system fully compatiblewith preexisting technologies for sample handling. For example, liquidsamples in labs are commonly transferred using volumetric pipettes.While there is some degree of standardization, variations in availabletip geometries could make compatibility with a custom interfacedifficult. However, given that these pipettes have been designed totransfer fluids into microcentrifuge tubes and microtiter plates, anycartridge with a sample input reservoir that mimics these other systemswill be at an advantage.

Further advantage can be gained through compatibility with standardparallel processing and automation equipment. Manual multipipettes androbotic pipetting systems are generally designed to interface with astandard 96-well microtiter plate format, i.e. with tips separated by a9 mm pitch and in arrangements of 8×12 (or a subset of this array). Thismakes cartridges that can nest together at a 9 mm pitch especiallyadvantageous.

Yet further ease of use can be achieved by making customer interactionwith the physical cartridge simple and obvious, especially whentroubleshooting. For example, transparency is a valuable though oftenoverlooked feature. A transparent sample input reservoir allows theoperator to confirm that the sample has been added to the cartridge, anda graduated reservoir allows confirmation that the necessary volume hasbeen added. A transparent fluid path allows operators to see if therehas been a problem with fluid handling or with bubbles.

Analyte Capture

While ease of use is important for broad acceptance by consumers, thekeystone of a diagnostic system is its detection technology. In onebroad class of such technologies, the specific binding of an analyte toan immobilized partner (commonly affixed to a surface) is the prelude toan observable signal. The rate at which such a capture event occurs isgenerally proportional to the concentration of analyte present at thatsurface.

As the analyte molecules nearest to the surface bind, the local analyteconcentration becomes depleted. This can slow the capture of subsequentmolecules, reducing the sensitivity of the system and thetime-to-answer, so efforts to replenish the analyte can reap rewards.For example, quickly flowing the analyte-containing fluid over thecapture site shrinks the boundary layer that bulk analyte molecules mustdiffuse across in order to be captured. In cases of extreme depletion,such fluid motion also carries new, “un-accessed” fractions of thesample nearer to the capture region.

There are many possible configurations for flowing a fluid across asurface. While not the only workable geometry, one convenientarrangement is to flow the analyte fluid through a channel over thecapture site. In this layout the constricted cross section increases theflow velocity for a given volume transferred, and several capturesurfaces can be placed sequentially along the channel to experience amore or less equivalent flow profile.

Actuating the motion of fluid through this channel can also be done inmany different ways. For a rectified flow, one configuration comprisesinserting a pump at one end of the system, or one in the middle with acheck valve on each side. Oscillating flow likely needs no valving butmight require a compression chamber if in a linear rather than cyclicalconfiguration.

One advantage to rectified flow that could justify its additionalcomplexity can be seen when considering bubbles, an additionalimpediment to analyte capture. If a capture surface is stored dry, it ispossible to generate bubbles of trapped air as the sample is introduced.Outgassing from the cartridge materials is also possible, and outgassingfrom the sample is virtually guaranteed given that the mixing techniquescommonly used to prepare samples will also aerate them. In each of thesescenarios, bubbles might reduce how effectively an analyte is captured.However, the ability to strip bubbles from the system can mitigate theissue. In a system with rectified flow, a bubble-stripping chamber canbe inserted into the fluid path.

For example, consider a design that could make use of the bubbles'buoyancy to extract them. Create a holding reservoir with an inlet thatadds fluid near the top and an outlet that removes it from the bottom.(Note that “top” and “bottom” do have a critical meaning in this contextbecause buoyancy is only meaningful in gravity or a similar massacceleration field.) Some reserve of standing fluid is present in thereservoir. When new fluid with bubbles enters from the top, the bubblesfloat on the top of this reserve until they collapse, while the newfluid mingles with the other liquid. Fluid is pulled from the bottom ofthis standing liquid to refresh the system.

It is important to design the system so that there is always standingliquid at the bottom of the reservoir, otherwise air or un-collapsedbubbles will get pulled from the chamber into the rest of the system anddefeat the purpose. (For example, there needs to be at least enoughliquid to accommodate any pulsing nature of the fluid propulsion.)However, there does not necessarily need to be any dead air space at thetop of the reservoir for this bubble-stripping chamber to work. Thechamber just needs to have a geometry such that the force due to thefluid flow pulling the liquid into the drain is less than the buoyancyforce propelling the bubbles away from the drain.

Electrochemical Detection

After an analyte has been captured, this binding event must be convertedinto a signal that is observable by a detection instrument. One way isto use electrochemistry to convert the chemical binding event into anelectrical current. In this embodiment, the capture surface must also bean electrode.

Electrochemical detection can have several advantages over alternativemethods, but it has its own special requirements. Electrochemicaltechniques call for the creation of an electrochemical cell, often athree-electrode cell where there is a working electrode (the sensorsurface) linked through a conductive electrolyte solution to anauxiliary electrode (a current source/sink) and a reference electrode (avoltage reference). Any disruption in this conductive link can impactthe electrochemical scan, so the bubble stripping described for analytecapture is just as important for electrochemical detection. The use ofredundant auxiliary electrodes is an additional way to reduce the riskof a break in conductivity: the path to each auxiliary electrode wouldhave to be blocked in order to compromise the electrochemical circuit.

Of course, electrical connectivity with the instrument is just asimportant as electrical connectivity through the electrolyte solution.Given the paradigm that many disposable cartridges will be interfacingwith a non-disposable piece of equipment, that equipment's connectormust be robust to many cycles. Furthermore that connector should be ableto create many connections in order to enable the analysis of manydifferent working electrodes from one cartridge. A zero insertion force(ZIF) connector with a two-dimensional grid of contacts is one goodsolution.

The following commonly owned or controlled patents describeelectrochemical detection principles and methodologies in more detail:U.S. Pat. Nos. 5,591,578, 5,824,473, 6,177,250, 6,277,576, 6,268,149,6,268,150, 6,180,352, 6,200,761 6,238,870, 6,258,545, 6,528,266,5,770,369, 6,096,273, 7,014,992, 6,221,583, 6,090,933, 7,045,285,6,479,240, 6,977,151, 7,125,668, 6,265,155, 6,291,188, 7,033,760,6,232,062, 6,495,323, 7,056,669, 6,013,459, 6,013,170, 6,248,229,7,018,523, 6,740,518, 6,063,573, 6,600,026, 7,160,678, 6,290,839,6,264,825, 6,761,816, 7,087,148, 6,541,617, 6,942,771, 6,432,723,6,833,267, 7,090,804, 6,686,150, 5,620,850, 6,197,515, 6,322,979,6,306,584, 7,172,897, 6,753,143, 6,518,024, 6,544,734, 6,642,046,6,592,696, 6,572,830, 6,361,958, 6,960,467, 6,602,400, 6,824,669,6,596,483, and 6,875,619, all of which are incorporated herein byreference for their disclosure related to detection principles andmethodologies.

Specific ligand attachment chemistries, including self-assemblingmonolayer technology, is also discussed in detail in U.S. Pat. Nos.6,306,584, 5,620,850, 6,472,148, 6,197,515, 6,322,979, 6,809,196,5,620,850, 6,197,515, 6,322,979, and 6,306,584, all of which areexpressly incorporated herein by reference for disclosure related toligand attachment chemistries, including self-assembling monolayertechnology (for example when the electrodes of the invention compriseself-assembled monolayers (SAMs)). The compositions of these SAMs willvary with the detection method used. In general, there are two basicdetection mechanisms. In one embodiment, detection of an ETM is based onelectron transfer through the stacked ni-orbitals of double strandednucleic acid. This basic mechanism is described in U.S. Pat. Nos.5,591,578, 5,770,369, 5,705,348, and PCT US97/20014 and is termed“mechanism-1” herein. Briefly, previous work has shown that electrontransfer can proceed rapidly through the stacked n-orbitals of doublestranded nucleic acid, and significantly more slowly throughsingle-stranded nucleic acid. Accordingly, this can serve as the basisof an assay. Thus, by adding ETMs (either covalently to one of thestrands or non-covalently to the hybridization complex through the useof hybridization indicators, described below) to a nucleic acid that isattached to a detection electrode via a conductive oligomer, electrontransfer between the ETM and the electrode, through the nucleic acid andconductive oligomer, may be detected.

Alternatively, the ETM can be detected, not necessarily via electrontransfer through nucleic acid, but rather can be directly detected on anelectrode comprising a SAM; that is, the electrons from the ETMs neednot travel through the stacked n orbitals in order to generate a signal.As above, in this embodiment, the detection electrode preferablycomprises a self-assembled monolayer (SAM) that serves to shield theelectrode from redox-active species in the sample. In this embodiment,the presence of ETMs on the surface of a SAM, that has been formulatedto comprise slight “defects” (sometimes referred to herein as“microconduits”, “nanoconduits” or “electroconduits”) can be directlydetected. This basic idea is termed “mechanism-2” herein. Essentially,the electroconduits allow particular ETMs access to the surface. Withoutbeing bound by theory, it should be noted that the configuration of theelectroconduit depends in part on the ETM chosen. For example, the useof relatively hydrophobic ETMs allows the use of hydrophobicelectroconduit forming species, which effectively exclude hydrophilic orcharged ETMs. Similarly, the use of more hydrophilic or charged speciesin the SAM may serve to exclude hydrophobic ETMs.

The person of ordinary skill in the art will appreciate thatelectrochemical detection may be accomplished in a variety of ways todetect a variety of different analytes. Osmetech's eSensor® DNADetection Technology is illustrative of one way of detecting nucleicacid sequences.

The eSensor® microarray is composed of a printed circuit board (PCB)consisting of an array of gold electrodes that are each modified with amulti-component, self-assembled monolayer (SAM) that includespre-synthesized oligonucleotide capture probes. Nucleic acid detectionis based on a sandwich assay principle. Signal and capture probes aredesigned with sequences complementary to immediately adjacent regions onthe corresponding target DNA sequence. A three-member complex is formedbetween capture probe, target, and signal probe based onsequence-specific hybridization, which brings the 5′-end of the signalprobe containing electrochemically-active ferrocene labels into closeproximity with the electrode surface. The ferrous ion within eachferrocene group undergoes cyclic oxidation and reduction, leading toloss or gain of an electron, which is measured as current at theelectrode surface using alternating current voltammetry (ACV) andhigher-order harmonic signal analysis. The ferrocene labels are onlydetected when the signal probe is captured at the surface of theelectrode by sequence-specific hybridization. In the absence of target,no specific signal is detected.

Osmetech's current system is adapted to specifically detect geneticmutations and polymorphisms by employing allele-specific signal probescontaining ferrocene labels with distinguishable redox potentials. Thesignal probe matching the wild-type sequence contains a ferrocene labelof one electrochemical potential, and a second signal probe matching themutant sequence contains a second, distinguishable ferrocene label. Boththe wild-type and mutant targets bind to the capture probe at a siteadjacent to the mutation. The wild-type and mutant signal probes thencompete for binding to their complementary sequences. The probe with theperfect match to the target is bound with a high degree of preference.The genotype is then determined by the ratio of signals generated by thebound wild-type and mutant signal probes. Genotyping boundaries areestablished based on statistical analysis of data from a large number ofsamples, and subsequent identification of unknown samples requires nofurther calibration of the instrument or cartridge lot. This approachcan be used to discriminate single- or multiple-base changes, insertionsand deletions. A mutation site with multiple alleles, or two adjacentmutation sites, can be genotyped using additional ferrocene labels.

Superior Cartridge/Instrument Interface

Regardless of the chosen analytical method, the interface betweencartridge and instrument must be designed for reliability andease-of-use. For example, appropriate alignment between the cartridgeand the fluid flow actuation must be ensured, keying features should beadded to prevent an operator from inserting a cartridge in the wrongorientation, and protective features may need to be added to preventdamage to the instrument if an inappropriate cartridge interface isforced. In addition, there should be tactile (and/or perhaps audible)feedback to the operator to communicate to them that a cartridge hasbeen inserted correctly. Common detent methods such as spring-loadedballs or compressible lever latches will serve this purpose.

It is also important to be forward-looking when designing thecartridge/instrument interface. Under the “disposable cartridge withreusable instrument” paradigm, new cartridges are frequently shipped tocustomers to be used with a device already installed in the field.Design choices can be made with that instrument's cartridge interfacesuch that it will be possible to upgrade cartridge design withouttriggering modification of the instruments themselves. For example, inone embodiment a pressure plate may press against the cartridge inintimate physical contact (to clamp that cartridge into place). However,if the pressure plate were to be designed to conform perfectly with thecartridge's natural design, then no other physical changes to thecartridge shape will be possible in the future without retrofit.However, if a mechanical adaptor is integrated into the cartridge sothat a more generic surface is presented to the pressure plate, thenfuture cartridge designs are free to change internal shape with acorresponding adjustment to the adaptor space within, the outside of thecartridge remaining substantially the same.

It should be noted that one or more of the above-noted preferredfeatures may not apply for a given application or embodiment, as theperson of ordinary skill in the art will appreciate.

EXAMPLES Example 1 Cartridge Design & Manufacture

With reference to FIG. 6, we have chosen a printed circuit board (PCB)(1) to provide the gold surfaces for a 3-electrode electrochemical cellcontrolled by a potentiostat circuit. It has dual auxiliary electrodes(3) shorted to one another, a multiplicity of working electrodes (4),and a reference electrode (5). The reference electrode is coated withthe same electrically conductive silver material (epoxy) that is used toattach an EEPROM (6), and this silver on the reference iselectrochemically oxidized immediately prior to analysis to generate thenecessary Ag/AgCl redox couple. (While a more standard solder could havebeen used to attach the EEPROM, this would have introduced additionalchemical compounds and manufacturing steps). The PCB geometry lays theworking electrodes in 3D wells, which simplifies the process of applyingdiffering chemical treatments to each electrode to create the desiredcapture surfaces.

In embodiment, the cartridge device consists of a PCB chip, a cover, anda microfluidic component. The microfluidic component is composed of aplate and a multilayer laminate. The PCB chip includes 72 gold-platedworking electrodes (that is twice density of the CFCD chip), asilver/silver chloride reference electrode, and two gold-platedauxiliary electrodes. Each working electrode has its own connectorcontact pad on the opposite side of the chip to allow electricalconnection to an instrument. The entire surface of PCB is coated with aninsulating solder mask, leaving only the center (250 mm diameter) of theelectrodes exposed. The PCB chip also contains an EEPROM (ElectricallyErasable-Programmable Read-Only Memory) component, a memory device thatstores information related to the cartridge, such as assay protocol,cartridge lot number, and expiration date.

With reference to FIGS. 1B, 2, 3, 4 and 5, a laminate assembly (2) isaffixed on top of the PCB (1), which assembly combines individual layersof pressure-sensitive adhesive (7 a-c) and thin plastic laminate layers(8 a,b). On top of the laminate assembly is a molded polycarbonate plate(9). The laminate layers define the fluid channel (10) and provide adiaphragm (11) (for interface with a pump) and two check valves. Thepolycarbonate plate (9) provides the rigid chimney (14), the extreme endof which constitutes the pump interface (15), and also contains a fillreservoir/sample chamber (16) that in this embodiment serves double dutyas a bubble-stripping chamber, but that in other embodiments may be aseparate chamber/port. All of this is created in a thin enougharrangement to allow cartridges to be held side-by-side in a loadingrack (17) with their reservoir openings (18) offset by a 9 mm standardmultipipette tip pitch or distance between individual cartridgechambers. For loading rack, see FIG. 10.

With reference to FIGS. 1B, 4 and 5, the walls of the fluid channel (10)are defined by one or more layers (7, 8) of the laminate assembly (2),and are sealed to the PCB (1) by an adhesive layer. While clamping theparts together might be sufficient in some systems, the adhesiveprovides a more reliable seal, especially for low viscosity solutions.(Although the conductive electrolyte solution necessary forelectrochemical detection is usually water-based, the specificadditional salts, detergents, or non-aqueous solvents required by theindividual chemistries of diagnostic technologies can have a significantimpact on the fluid's surface tension and capillary behavior.)

An adhesive layer can also seal the walls of the channel to its ceiling,which can be flat, beveled or otherwise. In one embodiment, the ceilingis formed from a plastic layer comprising a facing of the laminate orconforming channel within the laminate. This allows the laminate part tohold its shape during manufacture. Cutting the channel entirely throughto the polycarbonate plate could make the laminate prone to stretching,twisting, or distortion when handled, depending on the geometry of thechannel path. For this reason, in certain embodiments, certain of thelaminate layers are only partially cut into or sculpted, and notcompletely so, with other functionalities or complementing featuresprovided by other layers so that no one layer is too weak for practicalmanufacturing. In other embodiments, the ceiling is provided by thepolycarbonate plate/cover.

With reference to FIGS. 2, 4, 5, and 11, rectified fluid flow throughthe channel (10) is actuated by a diaphragm (11) flanked by two bridgebeams (13). The diaphragm (11) is formed from the same layer of plasticlaminate that creates the channel ceiling, covering an area where thechannel widens into a circular region. However, while the channelceiling is attached to the polycarbonate plate (9) with a layer ofadhesive, the diaphragm (11) is free to oscillate. A local absence ofadhesive and a recess/beveled underside (29) in the plate gives thediaphragm (11) freedom to flex toward and away from the PCB (1) surfaceand form a diaphragm chamber (22). See FIG. 11.

In one embodiment, the cartridge diaphragm (11) is driven by a pneumaticpump (12), preferably one integrated into or alongside a detectioninstrument/device (23). See FIGS. 13C, E. The use of alternatingpositive and negative pressure air pulses allows a full push-and-pullcycle, rather than the push-and-relax characteristic of other driversources. Furthermore, the pump interface (15) between the pneumatic pump(12) and cartridge (24) is quite simple. A flexible tube (25) carriesthe pressure wave, diverting it around any internal obstacles, anddelivers it to an outlet (26) configured for interface with acompressible grommet (27). Rigid chimney (14) molded into thepolycarbonate plate (9) mates with this opening and transfers the pulsethrough a narrowed baffle (28) and into beveled underside (29), which inturn mates in conforming fashion with cartridge diaphragm (11) uponsupply of a negative pressure/vacuum pulse from pump (12). See FIGS. 4,11.

With reference to FIGS. 4, 5, 8, and 11, rectified flow requiresdiaphragm pump zone to be communicatively flanked by a plurality ofcooperating check valves, here two. One embodiment incorporates what isreferred to as a “bridge” valves. Similar in concept to the common reedvalve, a bridge valve functions by seating a flat, flexible bridge beam(13) across a port (31) and valve seat (30) in a rigid or substantiallyrigid substrate. When a pressure differential pulls the fluid throughport (31), then flexible membrane (11) flexes away and allows fluid topass around it. When the direction of the pressure differentialreverses, the membrane gets pulled flat against the rigid valve seat andseals the port (31) against backwash. In one embodiment, the flexiblebridge beam (13) is formed from the same laminate layer (8 a) thatcreates the channel ceiling and diaphragm (11). The valve seats (30) arepreferably molded into the polycarbonate plate (9) and have slightlyraised annular bosses relative to membrane (11) that facilitate sealingwhen in closed position. The bridge valve has a simple construction thatdoes not require small floating parts or separate small pieces, whichleads to easier manufacture and greater reliability. The bridge valve ismore manufacturable even than its cousin, the reed valve. The “bridge”is constrained on both sides—on both “shores” if you will—whereas thereed would be attached on one side and free on the other (like a divingboard or flap), open to being bent or folded over during assembly (andthus subject to fatigue/weakening). The bridge valve has anotheradvantage over the reed valve in that it can be biased open, neutral, orclosed. This is controlled by the relative placement of the flexiblemembrane and the rigid valve seat. If the flat, relaxed bridge isexactly flush against the seat, the bias is neutral; if the relaxedbridge is separated from the seat, the bias is open; if the valve seatprotrudes so that the relaxed bridge is stretched and pressed againstit, the bias is closed. The common reed valve cannot be biased closed.]

One distinct feature of the cartridge over the CFCD cartridge is theutilization of microfluidic technology to introduce fluidic circulationin order to accelerate hybridization and decrease the hybridizationtime. The microfluidic component consists of a plastic plate and amulti-layer laminate sandwich, which form functional microfluidiccomponents such as a micropump, two check valves, and a hybridizationchannel. The laminate consists of multiple layers of silicone adhesive,Teflon, and a PET (polyethylene terephthalate) layer. The PET layerembodies two thin membranes in a bridge configuration that function ascheck valves and a diaphragm which is part of a pneumatically drivenpump. A serpentine channel (275 um deep and 1 mm wide) within thelaminate assembly forms the hybridization chamber above the workingelectrode array. The pneumatic pump is connected to a pneumatic sourcefrom the instrument. The pneumatic pump provides unidirectional pumpingof the analyte through the serpentine channel past all the electrodesduring the hybridization. Previous studies have shown that flowcirculation in the hybridization channel brought a large number oftarget molecules per time unit to pass by individual electrodes andallowed continuously replenishing the area around the electrode that hasbeen depleted of complementary targets. Moreover, since hybridization isa rate limiting process that relies on diffusion of target moleculesacross the diffusion boundary layer to their binding sites, the rapidfluid movements can enhance the transport of target within the diffusionboundary layer by reducing the thickness of the diffusion boundarylayer. As a result, the hybridization kinetics is greatly improved andhybridization time is reduced from 2 hr to 30 min.”

In operation, and with particular reference to FIGS. 2, 3, 4 and 11,fluid is introduced to the cartridge (24) by way of a filling reservoir(16) molded into the polycarbonate plate (9). The underside of theoutlet from this reservoir provides the valve seat (30) that togetherwith bridge beam (13) forms the first bridge valve. Fluid flows out ofthe reservoir (16), across this valve and into a diaphragm chamber (22).From the diaphragm chamber (22) fluid is diverted back up through thepolycarbonate plate (9) and across an overpass (34) so it can return tothe second bridge valve, exiting through a second valve seat (30) andtraversing a second bridge beam (13) to enter the primary channel (10).Fluid flows through this channel (10) over the electrodes (4) until itis released back into the filling/sample reservoir/chamber (16) where itis naturally stripped of bubbles before it begins another circuit. Ajumper laminate (35) seals the oval recess (36) in plate (9) associatedwith overpass (34) and second valve seat (30) and is planar andproximate to the ends of two substantially parallel ports (72), whichact to re-direct fluid from one plane to another in cartridge (24) andfacilitate rectified valve-actuated flow by taking advantage of gravity.Jumper laminate (35) conforms substantially to the dimension of the ovaloverpass recess (36) and is affixed by a jumper laminate pressuresensitive adhesive layer (33) of conforming dimension. See FIGS. 2, 3.Any dimension other than oval can be used, e.g., square, rectangular,circular. A separate molded polypropylene cap (38) with a living hinge(39) provides a seal for the filling reservoir. A rigid cover (40), alsomade from molded polycarbonate, fits over the top of the polycarbonateplate (9). It incorporates detents (41) to hold the PCB alignmentfeatures against their mating partners within the module (42) (discussedbelow) and to provide tactile and/or audible feedback. Cover (40) alsoincludes a handle (43) for easy manipulation in and out of module (42),keying features to ensure appropriate orientation before clamping thecartridge into the instrument, and clearance hole/recess (45) forreceipt of chimney (14) and for easy viewing of the filling/samplereservoir (16). A large, flat area (47) on the cover's surfaceinterfaces with the module clamping mechanism discussed below, andinternal ribs (underside of cover; not shown) transfer the pressureevenly across the polycarbonate plate (9). Space spanned by these ribsaffords adaptability for future inclusion of one or more additionalfunctionalities. Cover (40) is made of polycarbonate (injection-molded)and has a recess (49) to accommodate the fill reservoir/chamber (16) ofplate (9). The combined plate/assembly is attached to cover (40) by wayof two bosses (67) on plate that have reciprocal engagement recesses inthe underside of cover. See FIGS. 1A, B and 2. Each of laminate assembly(2), plate (9) and individual laminate layers (8) and adhesive layers(7) also have an EEPROM recess/void (70) for allowing access to theEEPROM (6).

The laminate assembly (2) and rigid plate (9) are both preferably madeof polyethyleneterepthalate (PET) and etched polytetrafluoroethylene(PTFE), and one or more of the individual laminate layers and PCB boardare held together by pressure sensitive adhesive (PSA) membranouslayers, preferably silicone-based. Other laminates that can be usedinclude but are not limited to, e.g., polycarbonate, polyethylene, ultrahigh molecular weight polyethylene (UHMW PE) and polypropylene. Criteriafor an acceptable laminate include one or more of flexibility (tensilemodulus), conformability (form by cold flow), pliability, ability tocut/machine or ablate (laser), ability to slice/make thin (form into afilm), durability, heat stability, and chemical inertness relative tothe fluidic components and assay chemistry therein. In addition, inplace of one or more laminates, silicone layers can be used that haveresilience/pliability and compressibility to facilitate sealing. Thematerials used for construction are readily available commercially:Adhesive layers of thicknesses 0.001-0.003″ from silicone pressuresensitive adhesives, for example, Tran-Sil NT1001 pressure sensitiveadhesive of thickness 0.002″ (Dielectric Polymers INC, Holyoke Mass.).Flexible materials such as skived Teflon® (PTFE) film of thickness0.002-0.010″ come from Fralock, Valencia Calif., and polycarbonate,polypropylene, and PET films 0.0005″-0.005″ thick come from NowPlastics, INC, East Longmeadow Mass. Pump specs: Micro Diaphragm, KNFNeuberger GmbH (Freiburg, DE), PU1947-NMP09-1.07, 6 Volts. These andother parts and materials are generic and commercially available orknown in the art, or else readily produced and assembled by the personof ordinary skill in the art with the guidance of the presentdisclosure.

The dimensions of the flow channel are significant in that the linearspeed of the fluid over the electrodes is dependent on the channel crosssection. Additionally, the channel height and width must be great enoughto allow any bubbles to freely flow through the channel and eventuallybe trapped/cleared in the reservoir. Therefore, it is important todesign a channel that is small enough to provide sufficient linearvelocity but not too restrictive with respect to bubble movement.Another consideration is that the lower channel dimensions will utilizeless analyte solution, often a desired characteristic. Channel widths of0.030-0.060″ and heights of 0.006″-0.0014″ were tested as compatibleranges, with the preferred dimensions being a width of 0.040″ and heightof 0.010″.

The overall channel dimensions directly impact the minimum amount offluid required to flow without lapses in analyte fluid (largebubbles/gaps) in the system. However, amounts in excess of the channelvolume can be cycled through the reservoir. The preferred system asdescribed above allows for analyte volumes of about 1100-200 uL,preferably about 130 uL or less. This range can be tuned easily byincreasing or decreasing the channel height via the thickness oflaminate assembly and contributing layers.

The dimensional aspects require exacting manufacturing methods such asthe use of laser cutting machinery instead of die cutting, as diecutting cannot form the thin channels (width of 0.030-0.040″) preferred.However, die-cutting could be used for laminates with features at theupper ends of the suggested ranges, e.g., 0.060″.

Material compatibility was thoroughly tested for Tran-Sil adhesives,with PET and PTFE Teflon® membrane components. The preferred laminateassembly consists of 0.003″ Transil® adhesive applied to the one side of0.006″ Teflon® (the side to be adhered to the PCB), another Tran-Sil®layer of 0.002″ thickness between the other side of the Teflon® the0.001″ PET middle laminate containing the bridge valves. The finaladhesive layer of 0.002″ Transil® is applied to the PET laminate andeventually bonds the polycarbonate cover to the laminate and PCB.

Example 2 Cartridge Module Design & Manufacture

With reference to FIGS. 13A-E, the interface between detection device(23) and cartridge (24) is mediated by a cartridge module (42), whichconsists of a base (50) and top (51), both of which are made of moldedpolyetherimide plastic (glass-filled Ultem®, General Electric), and bothof which contain aligned embedded metal screw castings (52) forfastening to each other. Module top (51) contains a connector lever (53)and engagement plate (54) for slideably engaging and disengagingcartridge firmly against base (50) and buckle beam assembly (73)electrical connection points therein. Top (51) also contains a conduit(55) for mating with the cartridge chimney (14) and feeding andwithdrawing air to and from said cartridge. Base (50) has a heater area(56) for receiving and mating with a gold/nickel plated copper thermalplate (57) and buckle beam assembly (73) that consists of an array ofgold/nickel plated beryllium copper pins parallel and proximate to saidheater area (56) for electrically engaging contact pads (58) beneath PCB(1) (see FIG. 7), and two spring-loaded stainless steel metal detentlocating pins (59) and radii (60) to aid final functional positioning ofcartridge upon initial positioning into module (42). Thermal plate (57)overlays heater area (56) and presses against the flat underside of thePCB (1) directly beneath cartridge fluid channel (10) to act as amodulable heat source for reactions taking place therein.

The electrical interface between device and cartridge relies onreciprocally cooperating features possessed by both. See FIGS. 7, 13A-D.In the PCB (1), traces (not shown) are routed from the electrodes (2-5)through vias (not shown) to the underside of the PCB (1), where thetwo-dimensional array of gold contact pads (58) are exposed to saidbuckle beam assembly (73), individual pads mating with individual pinsthereon. Module (42) is a clamping zero-insertion-force (ZIF) connectorwherein the cartridge is slotted in loose and then firmly engaged withinmodule (42) upon positionment against detent pins (59) and radii (60)and clamping with slideable lever (53) that forces engagement plate (54)down against top of cartridge (24), thereby securing bottom of cartridge(24), including pads (58), firmly against buckle beam assembly (73).Clamping motion also forcibly mates compressible outlet (63) ofpneumatic conduit (55) with cartridge chimney (14). Alignment features(64) cut into the PCB ensure that the array of pads (58) on underside ofthe PCB (1) and the array of pins from buckle beam assembly (73) lineup.

Buckle beam contacts can be substituted by pogo pins, anisotropicelectrically conductive tapes, films, elastomers, and or otherconnectors known in the art. Elastomeric parts for pneumatic interface,including compressible outlet, are made of molded polyurethane, butsilicone and like compositions are also considered to be an adequatealternative.

Example 3 Detection Device

In typical use, and with reference to FIG. 14, the cartridge andcartridge module are coupled to a detection device (23) and/or computer,which parts may be combined into one, as is known and readilyimplementable in the art. FIG. 14 shows an embodiment of a detectiondevice (23) for housing multiple cartridges (24). This embodimentcontains three vertical towers (65) each possessing eight cartridges(24), each of which fits into its own module (42). To the left is acomputer screen (66) for programming detection parameters and evaluatingresults. In this particular embodiment the screen is a touch-pad screenfrom which a user may conveniently select from various programmingoptions merely by touching the screen. The fill reservoir/chambers (16)of the cartridges (24) face out at an approximate 15° angle relative tohorizontal to take advantage of gravity and 3-dimensional space toeconomize/optimize onlooker or technician view and facilitateloading/clamping of cartridges into cartridge modules. For maximumutility, the detection device is programmed to perform either arandom-access or batch mode operation, which allows for one or moredifferent tests to be run simultaneously.

The preceding is useful for any detection device. Specifics ofelectrochemical detection and other detection methods are as describedpreviously and in the above-cited documents.

All articles and documents referenced herein, as well as all thecitations cited therein, are incorporated by reference for anunderstanding of the invention and are indicative of what the person ofordinary skill requires to know to make the invention operable using nomore than routine experimentation, as well as appreciate the advantagesof the invention.

One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The methodsand compositions described illustrate preferred embodiments, areexemplary, and are not intended as limitations on the scope of theinvention. Certain modifications and other uses will be apparent tothose skilled in the art, and are encompassed within the spirit of theinvention as defined by the scope of the claims.

The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention in the useof such terms and expressions of excluding any equivalents of thefeatures shown and described, or portions thereof. It is recognized thatvarious modifications are possible within the scope of the inventionclaimed. Thus, it should be understood that although the presentinvention has been specifically disclosed by preferred embodiments,optional features, modifications and variations of the concepts hereindisclosed may be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the description and the appended claims.

In addition, where features or aspects of the invention are described interms of ranges or Markush groups or other grouping of alternatives,e.g., genuses, those skilled in the art will recognize that theinvention is also thereby described in terms of any individualmeasurement, member or subgroup of members of the range, Markush groupor subgenus, and exclusions of individual members as appropriate, e.g.,by proviso.

1. A fluidics device comprising: a body, said body comprising; a flowchannel for transporting a fluid sample, said flow channel comprising asitus where an operation is performed on one or more components in saidfluid sample; an inlet port for receiving said fluid sample, said inletport in fluid communication with said flow channel and also incommunication with an enclosed gaseous environment; and an outlet portin fluid communication with said inlet port and configured such thatupon operation of said device said fluid sample is recirculated throughsaid flow channel to release gaseous bubbles into said enclosed gaseousenvironment while simultaneously allowing for facilitated diffusion andperformance of said operation on said one or more components in saidfluid sample.
 2. The fluidics device of claim 1 further comprising atleast one working electrode in said flow channel.
 3. The fluidics deviceof claim 2 further comprising one or more auxiliary electrodes.
 4. Thefluidics device of claim 1 further comprising connectors for interfacewith a detection device.
 5. The fluidics device of claim 1 furthercomprising an EEPROM.
 6. The fluidics device of claim 1 furthercomprising: one or more check valves in fluid communication with saidflow channel for regulating the flow of said fluid sample therethrough;and a diaphragm for interface with a pump, said diaphragm opearationallycoupled to said flow channel and said one or more check valves.
 7. Thefluidics device of claim 1 comprising one or more immobilized biologicalbinding partners or ligands in said flow channel.
 8. The fluidics deviceof claim 7 wherein said one or more biological binding partners orligands is immobilized using a self assembling monolayer affixed to oneor more electrode surfaces.
 9. The fluidics device of claim 1 comprisingan array of electrodes bound to one or more biological binding partnersor ligands.
 10. The fluidics device of claim 9 wherein said array islocated in a serpentine channel.
 11. The fluidics device of claim 1wherein said channel comprises a cross-sectional dimension comprising agreater width than height.
 12. The fluidics device of claim 1 whereinsaid device has a sample capacity of from about 50 ul to about 200 ul.13. A diagnostics kit comprising the device of fluidics device ofclaim
 1. 14. A method of determining analyte binding, comprising:providing a fluidics device according to claim 9; adding to said devicea liquid sample suspected of containing one or more analytes specificfor said one or more biological binding partners or ligands; circulatingand recirculating said sample across said array; and detecting bindingof said one or more analytes to said one or more biological bindingpartners or ligands.
 15. The method of claim 14 wherein said circulatingand recirculating comprises flowing said liquid sample at a rate of fromabout uL/sec to about 40 uL/sec.
 16. The fluidics device of claim 1wherein said device has a single fluid sample capacity of no more thanabout 200 uL in said flow channel and said flow channel has a length offrom about 100 mm to about 200 mm, a cross-sectional dimension rangingfrom about 0.75 mm to about 2.0 mm in width, and a height of from about0.125 mm to about 0.40 mm.